Publications by authors named "Emily Blyth"

36 Publications

Prophylactic antigen-specific T-cells targeting seven viral and fungal pathogens after allogeneic haemopoietic stem cell transplant.

Clin Transl Immunology 2021 15;10(3):e1249. Epub 2021 Mar 15.

Sydney Medical School University of Sydney Sydney NSW Australia.

Objectives: Adoptive immunotherapy using donor-derived antigen-specific T-cells can prevent and treat infection after allogeneic haemopoietic stem cell transplant (HSCT).

Methods: We treated 11 patients with a prophylactic infusion of 2 × 10 cells per square metre donor-derived T-cells targeting seven infections (six viral and one fungal) following HSCT. Targeted pathogens were cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, varicella zoster virus, influenza, BK virus (BKV) and .

Results: T-cell products were successfully generated in all patients with 10 products responsive to 6 or 7 infections. T-cell infusions were associated with increases in antigen-experienced activated CD8 T-cells by day 30. CMV, EBV and BKV reactivation occurred in the majority of patients and was well controlled except where glucocorticoids were administered soon after T-cell infusion. Three patients in that circumstance developed CMV tissue infection. No patient required treatment for invasive fungal infection. The most common CMV and EBV TCR clonotypes in the infusion product became the most common clonotypes seen at day 30 post-T-cell infusion. Donors and their recipients were recruited to the study prior to transplant. Grade III/IV graft-versus-host disease developed in four patients. At a median follow-up of 390 days post-transplant, six patients had died, 5 of relapse, and 1 of multi-organ failure. Infection did not contribute to death in any patient.

Conclusion: Rapid reconstitution of immunity to a broad range of viral and fungal infections can be achieved using a multi-pathogen-specific T-cell product. The development of GVHD after T-cell infusion suggests that infection-specific T-cell therapy after allogeneic stem cell transplant should be combined with other strategies to reduce graft-versus-host disease.
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http://dx.doi.org/10.1002/cti2.1249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7960021PMC
March 2021

Profiling the Blood Compartment of Hematopoietic Stem Cell Transplant Patients During Human Cytomegalovirus Reactivation.

Front Cell Infect Microbiol 2020 8;10:607470. Epub 2021 Jan 8.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

Human cytomegalovirus (HCMV) is a widespread pathogen establishing a latent infection in its host. HCMV reactivation is a major health burden in immunocompromised individuals, and is a major cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). Here we determined HCMV genomic levels using droplet digital PCR in different peripheral blood mononuclear cell (PBMC) populations in HCMV reactivating HSCT patients. This high sensitivity approach revealed that all PBMC populations harbored extremely low levels of viral DNA at the peak of HCMV DNAemia. Transcriptomic analysis of PBMCs from high-DNAemia samples revealed elevated expression of genes typical of HCMV specific T cells, while regulatory T cell enhancers as well as additional genes related to immune response were downregulated. Viral transcript levels in these samples were extremely low, but remarkably, the detected transcripts were mainly immediate early viral genes. Overall, our data indicate that HCMV DNAemia is associated with distinct signatures of immune response in the blood compartment, however it is not necessarily accompanied by substantial infection of PBMCs and the residual infected PBMCs are not productively infected.
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http://dx.doi.org/10.3389/fcimb.2020.607470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820775PMC
January 2021

Identification of SARS-CoV-2 Nucleocapsid and Spike T-Cell Epitopes for Assessing T-Cell Immunity.

J Virol 2021 02 24;95(6). Epub 2021 Feb 24.

Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia.

Developing optimal T-cell response assays to severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is critical for measuring the duration of immunity to this disease and assessing the efficacy of vaccine candidates. These assays need to target conserved regions of SARS-CoV-2 global variants and avoid cross-reactivity to seasonal human coronaviruses. To contribute to this effort, we employed an immunoinformatics analysis pipeline to identify immunogenic peptides resulting from conserved and highly networked regions with topological importance from the SARS-CoV-2 nucleocapsid and spike proteins. A total of 57 highly networked T-cell epitopes that are conserved across geographic viral variants were identified from these viral proteins, with a binding potential to diverse HLA alleles and 80 to 100% global population coverage. Importantly, 18 of these T-cell epitope derived peptides had limited homology to seasonal human coronaviruses making them promising candidates for SARS-CoV-2-specific T-cell immunity assays. Moreover, two of the NC-derived peptides elicited effector/polyfunctional responses of CD8 T cells derived from SARS-CoV-2 convalescent patients. The development of specific and validated immunologic tools is critical for understanding the level and duration of the cellular response induced by SARS-CoV-2 infection and/or vaccines against this novel coronavirus disease. To contribute to this effort, we employed an immunoinformatics analysis pipeline to define 57 SARS-CoV-2 immunogenic peptides within topologically important regions of the nucleocapsid (NC) and spike (S) proteins that will be effective for detecting cellular immune responses in 80 to 100% of the global population. Our immunoinformatics analysis revealed that 18 of these peptides had limited homology to circulating seasonal human coronaviruses and therefore are promising candidates for distinguishing SARS-CoV-2-specific immune responses from pre-existing coronavirus immunity. Importantly, CD8 T cells derived from SARS-CoV-2 survivors exhibited polyfunctional effector responses to two novel NC-derived peptides identified as HLA-binders. These studies provide a proof of concept that our immunoinformatics analysis pipeline identifies novel immunogens which can elicit polyfunctional SARS-CoV-2-specific T-cell responses.
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http://dx.doi.org/10.1128/JVI.02002-20DOI Listing
February 2021

Successful treatment of CMV, EBV, and adenovirus tissue infection following HLA-mismatched allogeneic stem cell transplant using infusion of third-party T cells from multiple donors in addition to antivirals, rituximab, and surgery.

Transpl Infect Dis 2020 Nov 24:e13528. Epub 2020 Nov 24.

Department of Haematology, Westmead Hospital, Sydney, NSW, Australia.

Viral infections, principally cytomegalovirus, Epstein Barr virus (EBV) and adenovirus, are a leading cause of morbidity and mortality after allogeneic stem cell transplantation. The use of systemic antivirals is limited by limited efficacy and organ toxicities. Inability to clear infection is exacerbated by transplant-related immunosuppression and prophylaxis or treatment of acute graft versus host disease. We report the first patient to clear three serious viral infections after stem cell transplant using third-party donor partially human leukocyte antigen (HLA) matched virus-specific cytotoxic T cells. The patient, a 53 year old female with transplanted for relapsed leukemia, with severe graft versus host disease received five T cell infusions from three separate donors that ultimately cleared serious systemic infections with cytomegalovirus and adenovirus, and an EBV-driven lymphoma. Systemic antivirals had resulted in failed clinical responses. Use of repeated infusions of partially HLA matched virus-specific T cells from banks containing cryopreserved cells should be strongly considered in transplant recipients with single or multiple refractory viral infections.
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http://dx.doi.org/10.1111/tid.13528DOI Listing
November 2020

enrichment of PRAME antigen-specific T cells for adoptive immunotherapy using CD137 activation marker selection.

Clin Transl Immunology 2020 21;9(10):e1200. Epub 2020 Oct 21.

Westmead Institute for Medical Research Westmead NSW Australia.

Objective: Adoptive immunotherapy with expanded tumor-specific T cells has potential as anticancer therapy. Preferentially expressed antigen in melanoma (PRAME) is an attractive target overexpressed in several cancers including melanoma and acute myeloid leukaemia (AML), with low expression in normal tissue outside the gonads. We developed a GMP-compliant manufacturing method for PRAME-specific T cells from healthy donors for adoptive immunotherapy.

Methods: Mononuclear cells were pulsed with PRAME 15-mer overlapping peptide mix. After 16 h, activated cells expressing CD137 were isolated with immunomagnetic beads and cocultured with irradiated CD137 fraction in medium supplemented with interleukin (IL)-2, IL-7 and IL-15. Cultured T cells were restimulated with antigen-pulsed autologous cells after 10 days. Cellular phenotype and cytokine response following antigen re-exposure were assessed with flow cytometry, enzyme-linked immunospot (ELISPOT) and supernatant cytokine detection. Detailed phenotypic and functional analysis with mass cytometry and T-cell receptor (TCR) beta clonality studies were performed on selected cultures.

Results: PRAME-stimulated cultures ( = 10) had mean expansion of 2500-fold at day 18. Mean CD3 percentage was 96% with CD4:CD8 ratio of 4:1. Re-exposure to PRAME peptide mixture showed enrichment of CD4 cells expressing interferon (IFN)-γ (mean: 12.2%) and TNF-α (mean: 19.7%). Central and effector memory cells were 23% and 72%, respectively, with 24% T cells expressing PD1. Mass cytometry showed predominance of Th1 phenotype (CXCR3/CCR4/CCR6/Tbet, mean: 73%) and cytokine production including IL-2, IL-4, IL-8, IL-13 and GM-CSF (2%, 6%, 8%, 4% and 11%, respectively).

Conclusion: PRAME-specific T cells for adoptive immunotherapy were enriched from healthy donor mononuclear cells. The products were oligoclonal, exhibited Th1 phenotype and produced multiple cytokines.
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http://dx.doi.org/10.1002/cti2.1200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7577233PMC
October 2020

Mass cytometry reveals immune signatures associated with cytomegalovirus (CMV) control in recipients of allogeneic haemopoietic stem cell transplant and CMV-specific T cells.

Clin Transl Immunology 2020 2;9(7):e1149. Epub 2020 Jul 2.

Faculty of Medicine and Health The University of Sydney Camperdown NSW Australia.

Objectives: Cytomegalovirus (CMV) is known to have a significant impact on immune recovery post-allogeneic haemopoietic stem cell transplant (HSCT). Adoptive therapy with donor-derived or third-party virus-specific T cells (VST) can restore CMV immunity leading to clinical benefit in prevention and treatment of post-HSCT infection. We developed a mass cytometry approach to study natural immune recovery post-HSCT and assess the mechanisms underlying the clinical benefits observed in recipients of VST.

Methods: A mass cytometry panel of 38 antibodies was utilised for global immune assessment (72 canonical innate and adaptive immune subsets) in HSCT recipients undergoing natural post-HSCT recovery ( = 13) and HSCT recipients who received third-party donor-derived CMV-VST as salvage for unresponsive CMV reactivation ( = 8).

Results: Mass cytometry identified distinct immune signatures associated with CMV characterised by a predominance of innate cells (monocytes and NK) seen early and an adaptive signature with activated CD8 T cells seen later. All CMV-VST recipients had failed standard antiviral pharmacotherapy as a criterion for trial involvement; 5/8 had failed to develop the adaptive immune signature by study enrolment despite significant CMV antigen exposure. Of these, VST administration resulted in development of the adaptive signature in association with CMV control in three patients. Failure to respond to CMV-VST in one patient was associated with persistent absence of the adaptive immune signature.

Conclusion: The clinical benefit of CMV-VST may be mediated by the recovery of an adaptive immune signature characterised by activated CD8 T cells.
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http://dx.doi.org/10.1002/cti2.1149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332355PMC
July 2020

Unrelated Donor Transplant Recipients Given Thymoglobuline Have Superior GRFS When Compared to Matched Related Donor Recipients Undergoing Transplantation without ATG.

Biol Blood Marrow Transplant 2020 10 5;26(10):1868-1875. Epub 2020 Jul 5.

Faculty of Medicine and Health, University of Sydney, Camperdown, New South Wales, Australia; Westmead Hospital, Westmead, New South Wales, Australia.

Recipients of allogeneic hematopoietic stem cell transplantation (HSCT) from unrelated donors (URDs) and mismatched related donors (MMRDs) typically have a higher incidence of acute and chronic graft-versus-host disease (GVHD) compared with matched related donors (MRDs). Anti-T-cell globulins (ATGs) are often used to reduce GVHD in these recipients. We report the outcomes of 211 adult peripheral blood stem cell transplant recipients with myeloid malignancies who received a standardized transplant protocol, in which ATG (Thymoglobuline 4.5 mg/kg) was administered to recipients of URD and MMRD (n = 147) but not MRD (n = 64) transplant. For all patients, incidence of acute GVHD grades 2 to 4 was 21.4%, and chronic GVHD was 35.0%. Two-year overall survival was 63.2% (95% confidence interval, 55.8% to 71.5%), relapse-free survival was 55.3% (47.4% to 64.6%), and GVHD-free, relapse-free survival (GRFS) was 30.7% (23.2% to 40.8%). There were no differences between recipients of MRDs and other donors in relapse, nonrelapse mortality, and overall and relapse-free survival. However, compared with MRD, recipients from URDs and MMRDs had reduced moderate to severe chronic GVHD (10.4% versus 30.1%, P= .002), less chronic GVHD requiring systemic therapy (19.4% versus 38.9%, P = .006), and superior 2-year GRFS (35.5% versus 20.0%, P = .003). In this retrospective review of nonrandomized transplant groups, outcomes of HSCT performed using an URD with ATG during conditioning were superior to transplant from an MRD without ATG. The addition of Thymoglobuline to conditioning in HSCT from MRD should be further examined in prospective trials.
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http://dx.doi.org/10.1016/j.bbmt.2020.06.030DOI Listing
October 2020

Whole-Genome Approach to Assessing Human Cytomegalovirus Dynamics in Transplant Patients Undergoing Antiviral Therapy.

Front Cell Infect Microbiol 2020 15;10:267. Epub 2020 Jun 15.

MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom.

Human cytomegalovirus (HCMV) is the most frequent cause of opportunistic viral infection following transplantation. Viral factors of potential clinical importance include the selection of mutants resistant to antiviral drugs and the occurrence of infections involving multiple HCMV strains. These factors are typically addressed by analyzing relevant HCMV genes by PCR and Sanger sequencing, which involves independent assays of limited sensitivity. To assess the dynamics of viral populations with high sensitivity, we applied high-throughput sequencing coupled with HCMV-adapted target enrichment to samples collected longitudinally from 11 transplant recipients (solid organ, = 9, and allogeneic hematopoietic stem cell, = 2). Only the latter presented multiple-strain infections. Four cases presented resistance mutations ( = 6), two (A594V and L595S) at high (100%) and four (V715M, V781I, A809V, and T838A) at low (<25%) frequency. One allogeneic hematopoietic stem cell transplant recipient presented up to four resistance mutations, each at low frequency. The use of high-throughput sequencing to monitor mutations and strain composition in people at risk of HCMV disease is of potential value in helping clinicians implement the most appropriate therapy.
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http://dx.doi.org/10.3389/fcimb.2020.00267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7308726PMC
June 2020

Managing haematology and oncology patients during the COVID-19 pandemic: interim consensus guidance.

Med J Aust 2020 06 13;212(10):481-489. Epub 2020 May 13.

Peter MacCallum Cancer Centre, Melbourne, VIC.

Introduction: A pandemic coronavirus, SARS-CoV-2, causes COVID-19, a potentially life-threatening respiratory disease. Patients with cancer may have compromised immunity due to their malignancy and/or treatment, and may be at elevated risk of severe COVID-19. Community transmission of COVID-19 could overwhelm health care services, compromising delivery of cancer care. This interim consensus guidance provides advice for clinicians managing patients with cancer during the pandemic.

Main Recommendations: During the COVID-19 pandemic: In patients with cancer with fever and/or respiratory symptoms, consider causes in addition to COVID-19, including other infections and therapy-related pneumonitis. For suspected or confirmed COVID-19, discuss temporary cessation of cancer therapy with a relevant specialist. Provide information on COVID-19 for patients and carers. Adopt measures within cancer centres to reduce risk of nosocomial SARS-CoV-2 acquisition; support population-wide social distancing; reduce demand on acute services; ensure adequate staffing; and provide culturally safe care. Measures should be equitable, transparent and proportionate to the COVID-19 threat. Consider the risks and benefits of modifying cancer therapies due to COVID-19. Communicate treatment modifications, and review once health service capacity allows. Consider potential impacts of COVID-19 on the blood supply and availability of stem cell donors. Discuss and document goals of care, and involve palliative care services in contingency planning.

Changes In Management As A Result Of This Statement: This interim consensus guidance provides a framework for clinicians managing patients with cancer during the COVID-19 pandemic. In view of the rapidly changing situation, clinicians must also monitor national, state, local and institutional policies, which will take precedence.

Endorsed By: Australasian Leukaemia and Lymphoma Group; Australasian Lung Cancer Trials Group; Australian and New Zealand Children's Haematology/Oncology Group; Australia and New Zealand Society of Palliative Medicine; Australasian Society for Infectious Diseases; Bone Marrow Transplantation Society of Australia and New Zealand; Cancer Council Australia; Cancer Nurses Society of Australia; Cancer Society of New Zealand; Clinical Oncology Society of Australia; Haematology Society of Australia and New Zealand; National Centre for Infections in Cancer; New Zealand Cancer Control Agency; New Zealand Society for Oncology; and Palliative Care Australia.
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http://dx.doi.org/10.5694/mja2.50607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7273031PMC
June 2020

Single cell analysis reveals human cytomegalovirus drives latently infected cells towards an anergic-like monocyte state.

Elife 2020 01 22;9. Epub 2020 Jan 22.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

Human cytomegalovirus (HCMV) causes a lifelong infection through establishment of latency. Although reactivation from latency can cause life-threatening disease, our molecular understanding of HCMV latency is incomplete. Here we use single cell RNA-seq analysis to characterize latency in monocytes and hematopoietic stem and progenitor cells (HSPCs). In monocytes, we identify host cell surface markers that enable enrichment of latent cells harboring higher viral transcript levels, which can reactivate more efficiently, and are characterized by reduced intrinsic immune response that is important for viral gene expression. Significantly, in latent HSPCs, viral transcripts could be detected only in monocyte progenitors and were also associated with reduced immune-response. Overall, our work indicates that regardless of the developmental stage in which HCMV infects, HCMV drives hematopoietic cells towards a weaker immune-responsive monocyte state and that this anergic-like state is crucial for the virus ability to express its transcripts and to eventually reactivate.
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http://dx.doi.org/10.7554/eLife.52168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039680PMC
January 2020

Pre- and post-bone marrow harvest anaemia is associated with lower CD34+ stem cell collection, high harvest volume and female gender.

Intern Med J 2020 03;50(3):299-306

Blood and Marrow Transplantation Program, Westmead Hospital, Sydney, New South Wales, Australia.

Background: Donor safety is paramount when performing bone marrow stem cell harvest. The incidence of full blood count (FBC) abnormalities among donors and variables associated with anaemia after marrow harvest are not well established.

Aims: To describe the frequency of FBC abnormalities prior to bone marrow stem cell harvest and to identify variables associated with post harvest anaemia.

Methods: Outcomes of 80 consecutive adult marrow harvests performed at our centre were analysed retrospectively.

Results: FBC abnormalities were present in 28% of donors prior to marrow harvest with normocytic anaemia the most common abnormality in 13%. Reduced donor haemoglobin (Hb) was independently correlated with lower CD34+ cell count per kg of recipient body weight. Anaemia (Hb < 100 g/L) was seen in 20% of donors after harvest with median decrease in Hb of 19 g/L. Variables independently associated with anaemia after harvest included donor to recipient weight ratio (P = 0.011), high collection volume (P = 0.044) and female gender (P = 0.023). Total nucleated cell and CD34 concentration in the final collected product were associated with the inverse of harvested marrow volume (P < 0.001).

Conclusions: Pre-harvest anaemia should be corrected where possible particularly in female donors. Marrow collection volume should be minimised to reduce post-harvest anaemia, optimise CD34+ cell number and improve nucleated and stem cell concentrations in the harvest product.
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http://dx.doi.org/10.1111/imj.14419DOI Listing
March 2020

Pathogen-Specific T Cells Beyond CMV, EBV and Adenovirus.

Curr Hematol Malig Rep 2019 08;14(4):247-260

Faculty of Medicine and Health, The University of Sydney, Camperdown, Australia.

Purpose Of Review: Infectious diseases contribute significantly to morbidity and mortality in recipients of allogeneic haematopoietic stem cell transplantation (aHSCT), particularly in the era of highly immunosuppressive transplant regimens and alternate donor transplants. Delayed cellular immune recovery is a major mechanism for the increased risk in these patients. Adoptive cell therapy with ex vivo manipulated pathogen-specific T cells (PSTs) is increasingly taking its place as a treatment strategy using donor-derived or third party-banked cells.

Recent Findings: The majority of clinical trial data in the form of early-phase studies has been in the prophylaxis or treatment of cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus (AdV). Advancements in methods to select and enrich PSTs offer the opportunity to target the less common viral pathogens as well as fungi with this technology. Early clinical studies of PSTs targeting polyomaviruses (BK virus and JC virus), human herpesvirus 6 (HHV6), varicella zoster virus (VZV) and Aspergillus spp. have shown promising results in small numbers of patients. Other potential targets include herpes simplex virus (HSV), respiratory viruses and other invasive fungal species. In this review, we describe the burden of disease of this wider spectrum of pathogens, the progress in the development of manufacturing capability, early clinical results and the opportunities and challenges for implementation in the clinic.
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http://dx.doi.org/10.1007/s11899-019-00521-zDOI Listing
August 2019

Human Cytomegalovirus Latency and Reactivation in Allogeneic Hematopoietic Stem Cell Transplant Recipients.

Front Microbiol 2019 28;10:1186. Epub 2019 May 28.

Discipline of Infectious Diseases and Immunology, Sydney Medical School, Charles Perkins Centre, University of Sydney, Sydney, NSW, Australia.

Human cytomegalovirus (HCMV) reactivation is a major infectious cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). HCMV is a ubiquitous beta-herpesvirus which asymptomatically infects immunocompetent individuals but establishes lifelong latency, with the potential to reactivate to a life-threatening productive infection when the host immune system is suppressed or compromised. Opportunistic HCMV reactivation is the most common viral complication following engraftment after HSCT and is associated with a marked increase in non-relapse mortality, which appears to be linked to complex effects on post-transplant immune recovery. This minireview explores the cellular sites of HCMV latency and reactivation in HSCT recipients and provides an overview of the risk factors for HCMV reactivation post-HSCT. The impact of HCMV in shaping post-transplant immune reconstitution and its relationship with patient outcomes such as relapse and graft-versus-host disease will be discussed. Finally, we survey current and emerging strategies to prevent and control HCMV reactivation in HSCT recipients, with recent developments including adoptive T cell therapies to accelerate HCMV-specific T cell reconstitution and new anti-HCMV drug therapy for HCMV reactivation after HSCT.
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http://dx.doi.org/10.3389/fmicb.2019.01186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546901PMC
May 2019

Restriction of Human Cytomegalovirus Infection by Galectin-9.

J Virol 2019 02 17;93(3). Epub 2019 Jan 17.

Discipline of Infectious Diseases and Immunology, University of Sydney, Camperdown, New South Wales, Australia

Human cytomegalovirus (HCMV) is a ubiquitous human herpesvirus. While HCMV infection is generally asymptomatic in the immunocompetent, it can have devastating consequences in those with compromised or underdeveloped immune systems, including transplant recipients and neonates. Galectins are a widely expressed protein family that have been demonstrated to modulate both antiviral immunity and regulate direct host-virus interactions. The potential for galectins to directly modulate HCMV infection has not previously been studied, and our results reveal that galectin-9 (Gal-9) can potently inhibit HCMV infection. Gal-9-mediated inhibition of HCMV was dependent upon its carbohydrate recognition domains and thus dependent on glycan interactions. Temperature shift studies revealed that Gal-9 specific inhibition was mediated primarily at the level of virus-cell fusion and not binding. Additionally, we found that during reactivation of HCMV in hematopoietic stem cell transplant (HSCT) patients soluble Gal-9 is upregulated. This study provides the first evidence for Gal-9 functioning as a potent antiviral defense effector molecule against HCMV infection and identifies it as a potential clinical candidate to restrict HCMV infections. Human cytomegalovirus (HCMV) continues to cause serious and often life-threatening disease in those with impaired or underdeveloped immune systems. This virus is able to infect and replicate in a wide range of human cell types, which enables the virus to spread to other individuals in a number of settings. Current antiviral drugs are associated with a significant toxicity profile, and there is no vaccine; these factors highlight a need to identify additional targets for the development of anti-HCMV therapies. We demonstrate for the first time that secretion of a member of the galectin family of proteins, galectin-9 (Gal-9), is upregulated during natural HCMV-reactivated infection and that this soluble cellular protein possesses a potent capacity to block HCMV infection by inhibiting virus entry into the host cell. Our findings support the possibility of harnessing the antiviral properties of Gal-9 to prevent HCMV infection and disease.
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http://dx.doi.org/10.1128/JVI.01746-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340044PMC
February 2019

Establishment and Operation of a Third-Party Virus-Specific T Cell Bank within an Allogeneic Stem Cell Transplant Program.

Biol Blood Marrow Transplant 2018 12 29;24(12):2433-2442. Epub 2018 Aug 29.

Westmead Institute for Medical Research, University of Sydney, Australia; Blood and Bone Marrow Transplant Unit, Westmead Hospital, Sydney, Australia; Department of Haematology, Westmead Hospital, Sydney, Australia; Sydney Cellular Therapies Laboratory, Westmead Hospital, Sydney, Australia; Sydney Medical School, University of Sydney, Sydney, Australia. Electronic address:

Hematopoietic stem cell transplantation (HSCT) donor-generated virus-specific T cells (VSTs) can provide effective treatment for viral infection post-HSCT but are not readily accessible to all patients. Off-the-shelf cryopreserved VSTs suitable for treatment of multiple patients are an attractive alternative. We generated a bank of 17 cytomegalovirus (CMV)-, 14 Epstein-Barr virus (EBV)-, and 15 adenovirus (AdV)-specific T cell products from 30 third-party donors. Donors were selected for expression of 6 core HLA antigens expressed at high frequency in the local transplant population. T cells were generated by co-culturing venous blood or mobilized hematopoietic stem cell (HSC)-derived mononuclear cells with monocyte-derived dendritic cells pulsed with overlapping peptides covering CMV pp65, AdV5 hexon, or EBV BZLF1/LMP2A/EBNA1 proteins. Addition of a CD14 selection step instead of plate adherence to isolate monocytes before culture initiation significantly improved expansion in cultures from HSC material. Phenotyping showed the CD8 subset to have significantly higher numbers of terminal effector T cells (CD45RA62L) and lower numbers of effector memory T cells (CD45RA62L) when compared with the CD4 subset. Increased expression of the immunoinhibitory markers PD-1 and TIM-3 was noted on CD4 but not CD8 cells when compared with the control group. VST showed antiviral activity restricted through a variety of common HLAs, and modelling suggested a suitably HLA-matched product would be available for >90% of HSCT patients. Only a small number of carefully selected third-party donors are required to generate a VST bank of broad coverage, indicating the feasibility of local banking integrated into existing allogeneic HSCT programs.
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http://dx.doi.org/10.1016/j.bbmt.2018.08.024DOI Listing
December 2018

Rescue haploidentical peripheral blood stem cell transplantation for engraftment failure: a single-centre case series.

Intern Med J 2018 08;48(8):988-991

Blood and Marrow Transplant Unit, Westmead Hospital, Sydney, New South Wales, Australia.

Graft failure affects approximately 5% of allogeneic stem cell transplants, with a poor prognosis. Salvage second allogeneic stem cell transplantation (alloSCT2) is limited by high rates of transplant-related mortality from infection and graft-versus-host disease. We report on five adult patients receiving rescue alloSCT2 using haploidentical peripheral blood stem cells. All patients achieved neutrophil engraftment, two subsequently died from sepsis and disease relapse, respectively. Three patients remain alive up to 2 years post-transplant. We suggest consideration of haploidentical alloSCT2 for patients with graft failure.
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http://dx.doi.org/10.1111/imj.13979DOI Listing
August 2018

Mass Cytometry for the Assessment of Immune Reconstitution After Hematopoietic Stem Cell Transplantation.

Front Immunol 2018 26;9:1672. Epub 2018 Jul 26.

University of Sydney, Sydney, NSW, Australia.

Mass cytometry, or Cytometry by Time-Of-Flight, is a powerful new platform for high-dimensional single-cell analysis of the immune system. It enables the simultaneous measurement of over 40 markers on individual cells through the use of monoclonal antibodies conjugated to rare-earth heavy-metal isotopes. In contrast to the fluorochromes used in conventional flow cytometry, metal isotopes display minimal signal overlap when resolved by single-cell mass spectrometry. This review focuses on the potential of mass cytometry as a novel technology for studying immune reconstitution in allogeneic hematopoietic stem cell transplant (HSCT) recipients. Reconstitution of a healthy donor-derived immune system after HSCT involves the coordinated regeneration of innate and adaptive immune cell subsets in the recipient. Mass cytometry presents an opportunity to investigate immune reconstitution post-HSCT from a systems-level perspective, by allowing the phenotypic and functional features of multiple cell populations to be assessed simultaneously. This review explores the current knowledge of immune reconstitution in HSCT recipients and highlights recent mass cytometry studies contributing to the field.
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http://dx.doi.org/10.3389/fimmu.2018.01672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070614PMC
September 2019

Ultra-Sensitive Droplet Digital PCR for the Assessment of Microchimerism in Cellular Therapies.

Biol Blood Marrow Transplant 2018 05 2;24(5):1069-1078. Epub 2018 Jan 2.

Westmead Institute of Medical Research, University of Sydney, Westmead, Australia; Sydney Medical School, University of Sydney, Camperdown, Australia; Sydney Cellular Therapies Laboratory, Westmead, Australia; Blood and Marrow Transplant Unit, Department of Haematology, Westmead Hospital, Westmead, Australia. Electronic address:

Current techniques to assess chimerism after hematopoietic stem cell transplantation (HSCT) are limited in both sensitivity and precision. These drawbacks are problematic in the context of cellular therapies that frequently result in microchimerism (donor chimerism <1%). We have developed a highly sensitive droplet digital PCR (ddPCR) assay using commercially available regents with good performance throughout the range of clinically relevant chimerism measurements, including microchimerism. We tested the assay using spiked samples of known donor-recipient ratios and in clinical samples from HSCT recipients and patients enrolled on clinical trials of microtransplantation and third-party virus-specific T cells (VSTs). The levels of detection and quantification of the assay were .008% and .023%, with high levels of precision with samples of DNA content ranging from 1 to 300 ng DNA. From the panel of 29 insertion-deletion probes multiple informative markers were found for each of 43 HSCT donor-recipient pairs. In the case of third-party cellular therapies in which there were 3 DNA contributors (recipient, HSCT donor, and T-cell donor), a marker to detect the cellular product in a background of recipient and donor cells was available for 11 of 12 cases (92%). Chimerism by ddPCR was able to quantify chimerism in HSCT recipients and comparison against standard STR analysis in 8 HSCT patients demonstrated similar results, with the advantage of fast turnaround time. Persistence of donor microchimerism in patients undergoing microtransplantation for acute myeloid leukemia was detectable for up to 57 days in peripheral blood and bone marrow. The presence of microtransplant product DNA in bone marrow T cells after cell sorting was seen in the 1 patient tested. In patients receiving third-party VSTs for treatment of refractory viral infections, VST donor DNA was detected at low levels in 7 of 9 cases. ddPCR offers advantages over currently available methods for assessment of chimerism in standard HSCT and cellular therapies.
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http://dx.doi.org/10.1016/j.bbmt.2017.12.802DOI Listing
May 2018

Long-term control of recurrent or refractory viral infections after allogeneic HSCT with third-party virus-specific T cells.

Blood Adv 2017 Nov 2;1(24):2193-2205. Epub 2017 Nov 2.

Westmead Institute for Medical Research, University of Sydney, Sydney, NSW, Australia.

Donor-derived adoptive T-cell therapy is a safe and effective treatment of viral infection posttransplant, but it is limited by donor serostatus and availability and by its personalized nature. Off-the-shelf, third-party virus-specific T cells (VSTs) appear promising, but the long-term safety and durability of responses have yet to be established. We conducted a prospective study of 30 allogeneic hemopoietic stem cell transplant (HSCT) patients with persistent or recurrent cytomegalovirus (CMV) (n = 28), Epstein-Barr virus (n = 1), or adenovirus (n = 1) after standard therapy. Patients were treated with infusions of partially HLA-matched, third-party, ex vivo-expanded VSTs (total = 50 infusions) at a median of 75 days post-HSCT (range, 37 to 349 days). Safety, viral dynamics, and immune recovery were monitored for 12 months. Infusions were safe and well tolerated. Acute graft versus host disease occurred in 2 patients, despite a median HLA match between VSTs and the recipient of 2 of 6 antigens. At 12 months, the cumulative incidence of overall response was 93%. Virological control was durable in the majority of patients; the reintroduction of antiviral therapy after the final infusion occurred in 5 patients. CMV-specific T-cell immunity rose significantly and coincided with a rise in CD8 terminal effector cells. PD-1 expression was elevated on CD8 lymphocytes before the administration of third-party T cells and remained elevated at the time of viral control. Third-party VSTs show prolonged benefit, with virological control achieved in association with the recovery of CD8 effector T cells possibly facilitated by VST infusion. This trial was registered at www.clinicaltrials.gov as #NCT02779439 and www.anzctr.org.au as #ACTRN12613000603718.
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http://dx.doi.org/10.1182/bloodadvances.2017010223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737128PMC
November 2017

Cellular therapy for multiple pathogen infections after hematopoietic stem cell transplant.

Cytotherapy 2017 11 15;19(11):1284-1301. Epub 2017 Sep 15.

Department of Haematology, Westmead Hospital, Sydney, Australia; The Westmead Institute for Medical Research, The University of Sydney, Sydney, Australia. Electronic address:

Hematopoietic stem cell transplantation (HSCT) represents the only crative treatment option for many hematological conditions but results in a profound T-cell deficiency in the post-HSCT period. Infections account for a significant proportion of non-relapse morbidity and mortality, and infections with multiple organisms either simultaneously or at different times after transplant are common. Adoptive cellular therapy (ACT) with prophylactic or therapeutic infusion of donor derived or third-party, pathogen-specific T-cells represents a novel methodology to rapidly reconstitute T-cell mediated immunity in this context. For cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infection, clear evidence of efficacy with limited toxicity has been observed, with response rates up to 90%. Infusion of third-party, partially human leukocyte antigen-matched pathogen-specific T-cells have also demonstrated remarkable efficacy with responses seen in up to 70% of patients with resistant CMV, EBV and adenoviral infection. This review addresses the nature of post-HSCT immune deficiency, the common infections that occur in the post-HSCT period and how advances in ACT manufacturing methodologies is allowing for wider implementation of T-cell therapies targeting multiple pathogens in HSCT recipients.
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http://dx.doi.org/10.1016/j.jcyt.2017.07.012DOI Listing
November 2017

Adjuvant Peptide Pulsed Dendritic Cell Vaccination in Addition to T Cell Adoptive Immunotherapy for Cytomegalovirus Infection in Allogeneic Hematopoietic Stem Cell Transplantation Recipients.

Biol Blood Marrow Transplant 2018 01 31;24(1):71-77. Epub 2017 Aug 31.

The Westmead Institute for Medical Research, The University of Sydney, Sydney, Australia; Blood and Marrow Transplant Unit, Westmead Hospital, Sydney, Australia; Sydney Cell and Gene Therapy Laboratory, Westmead Hospital, Sydney, Australia. Electronic address:

Adoptive cellular immunotherapy has been shown to be effective in the management of cytomegalovirus (CMV) reactivation and disease. Whether adjuvant dendritic cell (DC) vaccination will provide additional benefit in prophylaxis or treatment of CMV in hematoietic cell transplantation (HSCT) recipients is unknown. In this study, we administered prophylactic CMV-peptide specific T cell infusions, followed by 2 doses of intradermal CMV peptide-pulsed DC vaccine, to 4 HSCT recipients. There were no immediate adverse events associated with T cell infusion or DC vaccinations. One of the 4 patients developed grade III acute gut graft-versus-host disease. Immune reconstitution against CMV was detected in all 4 patients. Patients receiving CMV peptide-specific T cells and DC vaccination had peak immune reconstitution at least 10 days after the second DC vaccination. In summary, combining DC vaccine with T cell infusion appears feasible, although further study is required to ascertain its safety and efficacy in augmenting the effects of infusing donor-derived CMV-specific T cells.
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http://dx.doi.org/10.1016/j.bbmt.2017.08.028DOI Listing
January 2018

Herpes simplex virus type 1 (HSV-1) specific T-cell generation from HLA-A1- and HLA-A2-positive donors for adoptive immunotherapy.

Cytotherapy 2017 01 25;19(1):107-118. Epub 2016 Oct 25.

The Westmead Institute for Medical Research, Australia; Blood and Marrow Transplant Unit, Australia; Sydney Cell and Gene Therapy Laboratory, Westmead Hospital, The University of Sydney, Sydney, Australia. Electronic address:

Background Aims: Herpes simplex virus (HSV) reactivation and infection is common in patients undergoing hematopoietic stem cell transplant (HSCT) and requires routine antiviral prophylaxis. Drug-resistant strains are increasingly common, and effective alternative therapy is currently unavailable. We generated and characterized HSV-1-specific T cells for use in adoptive cellular immunotherapy following allogeneic stem cell transplantation.

Methods: Peripheral blood mononuclear cells from HLA-A1 and HLA-A2 HSV-seropositive hereditary hemochromatosis donors were used as the antigen source. Three HLA-A1 and four HLA-A2 specific epitopes were used for stimulation of T cells. Cells were stimulated with antigen-pulsed dendritic cells and cultured for 21 days in medium with interleukin (IL)-2. Cultured cells were phenotyped and tested for cytokine production, proliferation and cytotoxicity.

Results: There was a 5.3-fold expansion in total cell numbers over 21 days of culture, with 35% of T cells being CD8 positive. Thirty-five percent, 21% and 5% of CD8 cells secreted interferon-γ, tumor necrosis factor-α and IL-2 upon HSV antigen re-stimulation. More than 50% of antigen-specific T cells secreted multiple cytokines. Cultured T cells proliferated upon antigen re-stimulation and lysed HSV-1 peptide and virus-infected targets.

Conclusions: It is feasible to generate functional HSV-1 specific T cells from the blood of HLA-A1 and HLA-A2 HSV-seropositive donors using specific peptides. The utility of these cells in preventing and treating HSV-1 reactivation in allogeneic HSCT will need to be tested clinically.
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http://dx.doi.org/10.1016/j.jcyt.2016.09.013DOI Listing
January 2017

CMV-specific immune reconstitution following allogeneic stem cell transplantation.

Virulence 2016 11 9;7(8):967-980. Epub 2016 Aug 9.

a Westmead Institute for Medical Research at the University of Sydney , Westmead , Sydney , Australia.

Cytomegalovirus (CMV) remains a major contributor to morbidity and mortality following allogeneic haemopoietic stem cell transplant (HSCT) despite widespread use of viraemia monitoring and pre-emptive antiviral therapy. Uncontrolled viral replication occurs primarily in the first 100 d post transplant but this high risk period can extend to many months if immune recovery is delayed. The re-establishment of a functional population of cellular effectors is essential for control of virus replication and depends on recipient and donor serostatus, the stem cell source, degree of HLA matching and post-transplant factors such as CMV antigen exposure, presence of GVHD and ongoing use of immune suppression. A number of immune monitoring assays exist but have not yet become widely accessible for routine clinical use. Vaccination, adoptive transfer of CMV specific T cells and a number of graft engineering processes are being evaluated to enhance of CMV specific immune recovery post HSCT.
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http://dx.doi.org/10.1080/21505594.2016.1221022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5160403PMC
November 2016

A fatal case of acute HHV-6 myocarditis following allogeneic haemopoietic stem cell transplantation.

J Clin Virol 2015 Nov 9;72:82-4. Epub 2015 Oct 9.

Department of Haematology, Westmead Hospital, Sydney, Australia; The Westmead Millennium Institute for Medical Research, The University of Sydney, Sydney, Australia; Sydney Medical School, The University of Sydney, Sydney, Australia. Electronic address:

Human herpesvirus 6 (HHV-6) is an ubiquitous virus that can reactivate in immunocompromised hosts, resulting in diverse clinical sequelae. We describe a case of fatal acute HHV-6 myocarditis in a patient who underwent allogeneic haemopoietic stem cell transplantation (HSCT). To our knowledge, this is the first reported case of biopsy proven HHV-6 myocarditis post-HSCT.
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http://dx.doi.org/10.1016/j.jcv.2015.09.013DOI Listing
November 2015

Addition of varicella zoster virus-specific T cells to cytomegalovirus, Epstein-Barr virus and adenovirus tri-specific T cells as adoptive immunotherapy in patients undergoing allogeneic hematopoietic stem cell transplantation.

Cytotherapy 2015 Oct;17(10):1406-20

Faculty of Medicine, University of Sydney, Sydney, Australia; Westmead Millennium Institute, Centre for Cancer Research, Sydney, Australia; Blood and Marrow Transplant Unit, Department of Haematology, Westmead Hospital, Sydney, Australia; Sydney Cell and Gene Therapy Laboratory, Westmead Hospital, Sydney, Australia. Electronic address:

Background Aims: Virus-specific T-cell immunotherapy is emerging as a promising management strategy for virus infections in patients after hematopoietic stem cell transplant (HSCT). Here we present outcomes of 10 adult patients who received multi-virus-specific T cells prophylactically after HSCT.

Methods: Donor-derived cytomegalovirus (CMV)-, Epstein-Barr virus (EBV)-, adenoviral- and varicella zoster virus (VZV)-specific T cells were generated in a single culture and administered to HSCT patients at a dose of 2 × 10(7)/m(2) virus-specific T cells at a median of 63 days post-transplant. Patients were monitored for 12 months for evidence of viral reactivation and graft-versus-host disease.

Results: There was no acute infusion-related toxicity. Six patients developed CMV reactivation after T-cell infusion with a median peak CMV DNA titer of 600 copies per milliliter, and 1 received CMV-specific pharmacotherapy post-infusion. No EBV, adenoviral or VZV reactivation or disease was reported. Using interferon-γ Elispot analysis on post-infusion samples, we identified anti-viral immunity against all viruses including VZV. Three patients (30%) developed grade II-IV acute graft-versus-host disease.

Conclusions: This is the first description of the use of a multi-virus-specific T-cell product containing cells specific for VZV after allogeneic HSCT. The T-cell product appears safe in the setting of HSCT and confirms our previous findings regarding CMV control and treatment. A larger study with longer follow-up is required to determine the efficacy of VZV-specific T cells given prophylactically in controlling episodes of herpes zoster and disseminated varicella infection after cessation of prophylactic anti-viral treatment.
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http://dx.doi.org/10.1016/j.jcyt.2015.07.005DOI Listing
October 2015

Low-cost generation of Good Manufacturing Practice-grade CD19-specific chimeric antigen receptor-expressing T cells using piggyBac gene transfer and patient-derived materials.

Cytotherapy 2015 Sep 23;17(9):1251-67. Epub 2015 Jul 23.

The Westmead Millennium Institute for Medical Research, The University of Sydney, Westmead, Australia; The Department of Haematology, Westmead Hospital, Westmead, Australia; Sydney Cellular Therapies Laboratory, Pathology West, Westmead, Australia. Electronic address:

Background Aims: Protocols for the production of CD19-specific chimeric antigen receptor (CAR19) T cells are often complex and expensive because of the use of retroviral and lentiviral vectors or the need for CAR19 T-cell enrichment. We aimed to simplify the generation of CAR19 T cells from the peripheral blood of normal donors and patients using the piggyBac transposon system of gene modification.

Methods: We varied electroporation voltage, cytokines and stimulation conditions for the generation and expansion of CAR19 T cells over a 3-week culture period.

Results: Using optimized electroporation voltage, interleukin-15 alone and co-culturing CAR T cells with peripheral blood mononuclear cells, we were able to expand CAR19 T-cell cultures by up to 765-fold over 3 weeks in normal donors and 180-fold in patients with B-cell malignancies. Final median CAR19 expression of 72% was seen in normal donors, and 81% was seen in patients with acute lymphoblastic leukaemia, chronic lymphocytic leukemia or non-Hodgkin lymphoma. CAR19 T cells produced interferon gamma on stimulation with CD19(+) cell lines and efficiently lysed both CD19(+) cell lines and primary leukemia cells. In addition, combining CAR expression with an inducible caspase safety switch allowed elimination of CAR19 T cells by the application of a small molecule dimerizer.

Discussion: We have produced a simple, inexpensive and easily adoptable protocol for the generation of CAR19 T cells suitable for use in clinical trials using the piggyBac transposon system. This provides a robust platform for further enhancing the T-cell product and testing new CAR technologies.
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http://dx.doi.org/10.1016/j.jcyt.2015.05.013DOI Listing
September 2015

Influence of Stem Cell Source on Outcomes of Allogeneic Reduced-Intensity Conditioning Therapy Transplants Using Haploidentical Related Donors.

Biol Blood Marrow Transplant 2015 Sep 14;21(9):1641-5. Epub 2015 Jun 14.

Blood and Marrow Transplant Service, Haematology Department, Westmead Hospital, Westmead, New South Wales, Australia.

We compared outcomes for 2 retrospective cohorts of patients undergoing reduced-intensity conditioning (RIC) therapy transplants using haploidentical related donors and post-transplant prophylaxis against graft-versus-host disease (GVHD) with high-dose cyclophosphamide, tacrolimus, and mycophenolate. The first cohort of 13 was transplanted with bone marrow (BM) as the stem cell source, whereas the second cohort of 23 used peripheral blood stem cells (PBSCs) mobilized with granulocyte colony-stimulating factor. The BM cohort received a single 60-mg/kg dose of cyclophosphamide on day +3, whereas the PBSC cohort received 2 doses on days +3 and +4. Patients in the first cohort were slightly older and had a higher proportion of acute myeloid leukemia, but there were no differences in the distribution of Disease Risk Index scores between the 2 groups. Patients in the PBSC group received double the number of CD34(+) cells in the stem cell graft. Times to neutrophil and platelet recovery were not different between the 2 groups. Three patients, all in the PBSC group, failed to engraft but recovered with autologous hemopoiesis and survived. The 6-month cumulative incidences of acute GVHD were 55.1% for BM and 48.5% for PBSCs (P = .651), whereas 24-month cumulative rates for chronic GHVD were 28.6% for BM and 32.3% for PBSCs (P = .685). Only 2 patients, both in the BM group, died of nonrelapse causes, both of second cancers. The 2-year cumulative incidences of relapse were 43.9% for BM and 23.5% for PBSCs (P = .286). Overall survival at 2 years was significantly better for PBSC patients (P = .028), at 83.4% versus 52.7% for BM. Relapse-free and event-free survival did not differ significantly between BM and PBSC groups. In this retrospective analysis, we conclude that the use of PBSCs for haploidentical RIC transplants is a feasible strategy, with equivalent rates of acute and chronic GVHD and risk of relapse and low nonrelapse mortality compared with BM.
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http://dx.doi.org/10.1016/j.bbmt.2015.06.006DOI Listing
September 2015

Single-Agent High-Dose Cyclophosphamide for Graft-versus-Host Disease Prophylaxis in Human Leukocyte Antigen-Matched Reduced-Intensity Peripheral Blood Stem Cell Transplantation Results in an Unacceptably High Rate of Severe Acute Graft-versus-Host Disease.

Biol Blood Marrow Transplant 2015 May 27;21(5):941-4. Epub 2015 Jan 27.

BMT Service, Westmead Hospital, Sydney, New South Wales, Australia.

High-dose cyclophosphamide given early after allogeneic hemopoietic cell transplantation has been shown to be effective prophylaxis against graft-versus-host disease (GVHD) in the setting of HLA-matched myeloablative bone marrow grafts, allowing avoidance of long-term immunosuppression with calcineurin inhibitors in some patients. Whether this approach is feasible using granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cell grafts is unknown. We conducted an exploratory phase 2 trial of cyclophosphamide given at 50 mg/kg i.v. on days 3 and 4 after transplantation as sole GVHD prophylaxis in recipients of G-CSF-mobilized peripheral blood stem cell grafts from HLA-matched related or unrelated donors after reduced-intensity conditioning therapy with fludarabine, carmustine, and melphalan. Five patients, ages 52 to 67 years, with high-risk hematologic malignancies were enrolled. Four of the 5 developed severe acute GVHD of grades 3 to 4, requiring treatment with methylprednisolone and cyclosporine; 3 were steroid refractory and were given salvage therapy. One of these 4 patients died of hepatic GVHD, one died of sepsis, and 2 survived. We conclude that post-transplantation cyclophosphamide is inadequate as sole GVHD prophylaxis in the context of peripheral blood reduced-intensity conditioning transplantations from HLA-matched donors. This trial is registered at ACTRN12613001154796.
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http://dx.doi.org/10.1016/j.bbmt.2015.01.020DOI Listing
May 2015

Donor-derived CMV-specific T cells reduce the requirement for CMV-directed pharmacotherapy after allogeneic stem cell transplantation.

Blood 2013 May 22;121(18):3745-58. Epub 2013 Feb 22.

Westmead Millennium Institute, University of Sydney, Sydney, Australia.

We investigated the use of adoptively transferred donor-derived cytomegalovirus (CMV) specific cytotoxic T lymphocytes (CTL) as immune reconstitution postallogeneic transplant in a phase 2 study. Fifty patients were infused with a single dose of 2 × 10(7)cells/m(2) after day 28 post-transplant. Twenty-six patients reactivated CMV posttransplant (only 5 post-CTL infusion) and 9 required therapy with ganciclovir or foscarnet (only 1 post-CTL infusion). There was 1 case of fatal CMV disease, attributable to high levels of antithymocyte globulin at the time of T cell infusion. We compared the patients in the phase 2 study with a group of contemporaneous controls also treated at the trial centers. There was no increase in acute or chronic graft-versus-host disease attributable to CTL infusion; overall and progression-free survival were similar in both groups. There was a reduction in the percentage of patients who required CMV directed antiviral therapy (17% vs 36%, P = .01) and in the total number of treatment days in the cohort receiving CTL (3.4 days vs 8.9 days, P = .03) without a reduction in CMV reactivation rates. We postulate that adoptively transferred cells are able to expand in response to viral antigen, limit viral replication, and prevent progression to tissue infection. This study was registered on the Australian Clinical Trial Registry as #ACTRN12605000213640 and #ACTRN12607000224426.
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http://dx.doi.org/10.1182/blood-2012-08-448977DOI Listing
May 2013

Cytomegalovirus-specific cytotoxic T lymphocytes can be efficiently expanded from granulocyte colony-stimulating factor-mobilized hemopoietic progenitor cell products ex vivo and safely transferred to stem cell transplantation recipients to facilitate immune reconstitution.

Biol Blood Marrow Transplant 2013 May 1;19(5):725-34. Epub 2013 Feb 1.

Westmead Millennium Institute, Westmead Institute for Cancer Research, University of Sydney, Westmead, Australia.

Uncontrolled cytomegalovirus (CMV) reactivation after allogeneic hematopoietic stem cell transplantation causes significant morbidity and mortality. Adoptive transfer of CMV-specific cytotoxic T lymphocytes (CTLs) is a promising therapy to treat reactivation and prevent viral disease. In this article, we describe the generation of clinical-grade CMV-specific CTLs directly from granulocyte colony-stimulating factor-mobilized hemopoietic progenitor cell (G-HPC) products collected for transplantation. This method requires less than 2.5% of a typical G-HPC product to reproducibly expand CMV-specific CTLs ex vivo. Comparison of 11 CMV CTL lines generated from G-HPC products with 52 CMV CTL lines generated from nonmobilized peripheral blood revealed similar expansion kinetics and phenotype. G-HPC-derived CTLs produced IFN-γ after reexposure to CMVpp65 antigen and exhibited CMV-directed cytotoxicity but no alloreactivity against transplantation recipient-derived cells. Seven patients received CMV-specific CTL lines expanded from G-HPC products in a prophylactic adoptive immunotherapy phase I/II clinical trial. Use of G-HPC products will facilitate integration of CTL generation into established quality systems of transplantation centers and more rapid inclusion of T cell therapies into routine clinical care.
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http://dx.doi.org/10.1016/j.bbmt.2013.01.021DOI Listing
May 2013