Stuart Adams - Academia.edu (original) (raw)
Papers by Stuart Adams
Pediatric Transplantation, 2020
Paediatric heart transplantation recipients suffer an increased incidence of infectious, autoimmu... more Paediatric heart transplantation recipients suffer an increased incidence of infectious, autoimmune and allergic problems. The relative roles of thymus excision and immunosuppressive treatments in contributing to these sequelae are not clear. We compared the immunological phenotypes of 25 heart transplant recipients (Tx), 10 children who underwent thymus excision during non‐transplantation cardiac surgery (TE) and 25 age range–matched controls, in two age bands: 1‐9 and 10‐16 years. Significant differences from controls were seen mainly in the younger age band with Tx showing lower CD3 and CD4 cell counts whilst TE showed lower CD8 cell counts. Naïve T cell and recent thymic emigrant proportions and counts were significantly lower than controls in both groups in the lower age band. T cell recombination excision circle (TREC) levels were lower than controls in both groups in both age bands. There were no differences in regulatory T cells, but in those undergoing thymus excision in infancy, their proportions were higher in TE than Tx, a possible direct effect of immunosuppression. T cell receptor V beta spectratyping showed fewer peaks in both groups than in controls (predominantly in the older age band). Thymus excision in infancy was associated with lower CD8 cell counts and higher proportions of Tregs in TE compared to Tx. These data are consistent with thymus excision, particularly in infancy, being the most important influence on immunological phenotype after heart transplantation.
Blood
Historically, survival and activity of individual human hematopoietic progenitor subtypes have be... more Historically, survival and activity of individual human hematopoietic progenitor subtypes have been studied exclusively via transplantation in permissive mouse models. Despite being a relevant investigational tool, such animal models do not recapitulate the human hematopoietic milieu. Specifically, the production of human T and NK cells in these mice is severely impaired therefore, as of today, it has been impossible to measure with enough confidence the in vivo dynamics of human T/NK lymphoid progenitors. Hematopoietic stem cell gene therapy (GT) using integrating viral vectors has opened a unique opportunity to trace the fate of transplanted cells for the first time directly in vivo in humans by means of integration sites (IS) clonal tracking. Our mathematical modelling of IS data in clinical GT supported the existence of a population of long-term lymphoid progenitors (LtLP) capable of surviving independently from hematopoietic stem cells (HSC). However, to date, no experimental s...
Frontiers in Immunology
It is intriguing that, unlike adults with HIV-1, children with HIV-1 reach a greater CD4+ T cell ... more It is intriguing that, unlike adults with HIV-1, children with HIV-1 reach a greater CD4+ T cell recovery following planned treatment cessation. The reasons for the better outcomes in children remain unknown but may be related to increased thymic output and diversity of T cell receptor repertoires. HIV-1 infected children from the PENTA 11 trial tolerated planned treatment interruption without adverse long-term clinical, virological, or immunological consequences, once antiretroviral therapy was re-introduced. This contrasts to treatment interruption trials of HIV-1 infected adults, who had rapid changes in T cells and slow recovery when antiretroviral therapy was restarted. How children can develop such effective immune responses to planned treatment interruption may be critical for future studies. PENTA 11 was a randomized, phase II trial of planned treatment interruptions in HIV-1-infected children (ISRCTN 36694210). In this sub-study, eight patients in long-term follow-up were c...
Science Translational Medicine, 2011
X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the common cytokine... more X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the common cytokine receptor g chain. These mutations classically lead to complete absence of functional T and natural killer cell lineages as well as to intrinsically compromised B cell function. Although human leukocyte antigen (HLA)-matched hematopoietic stem cell transplantation (HSCT) is highly successful in SCID-X1 patients, HLA-mismatched procedures can be associated with prolonged immunodeficiency, graft-versus-host disease, and increased overall mortality. Here, 10 children were treated with autologous CD34+ hematopoietic stem and progenitor cells transduced with a conventional gammaretroviral vector. The patients did not receive myelosuppressive conditioning and were monitored for immunological recovery after cell infusion. All patients were alive after a median follow-up of 80 months (range, 54 to 107 months), and a functional polyclonal T cell repertoire was restored in all patients. Humoral im...
Blood Advances
The action of hematopoietic cell transplantation in controlling leukemia is principally mediated ... more The action of hematopoietic cell transplantation in controlling leukemia is principally mediated by donor T cells directed against residual recipient malignant cells. However, its utility is limited by graft-versus-host disease (GVHD), where alloreactivity is extended beyond leukemic and marrow cells. In a human/murine chimeric model, we previously showed that the preferential infiltration of cord blood (CB) CD8+ T cells eradicates an Epstein-Barr virus–driven lymphoblastoid tumor without causing xenogeneic GVHD. In the clinic, however, cord blood CD8+ T-cell reconstitution is significantly delayed, and the observation of such a robust antileukemia effect mediated by cord blood CD8+ T cells has not been reported. We describe an observation of very early T-cell expansion in 4 high-risk pediatric leukemia patients receiving third-party, pooled granulocytes after T cell–replete CB transplantation (CBT). The T-cell expansion was transient but robust, including expansion of CD8+ T cells,...
International Journal of Neonatal Screening
Spinal muscular atrophy (SMA) is an autosomal inherited neuromuscular genetic disease caused, in ... more Spinal muscular atrophy (SMA) is an autosomal inherited neuromuscular genetic disease caused, in 95% of cases, by homozygous deletions involving the SMN1 gene exon 7. It remains the leading cause of death in children under 2 years of age. New treatments have been developed and adopted for use in many countries, including the UK. Success of these treatments depends on early diagnosis and intervention in newborn babies, and many countries have implemented a newborn screening (NBS) or pilot NBS program to detect SMN1 exon 7 deletions on dried blood spots. In the UK, there is no current NBS program for SMA, and no pilot studies have commenced. For consideration of adoption of NBS for a new condition, numerous criteria must be satisfied, including critical assessment of a working methodology. This study uses a commercially available real-time PCR assay to simultaneously detect two different DNA segments (SMN1 exon 7 and control gene RPP30) using DNA extracted from a dried blood spot. Thi...
Journal of Clinical Immunology
Background Improved survival in ADA-SCID patients is revealing new aspects of the systemic disord... more Background Improved survival in ADA-SCID patients is revealing new aspects of the systemic disorder. Although increasing numbers of reports describe the systemic manifestations of adenosine deaminase deficiency, currently there are no studies in the literature evaluating genital development and pubertal progress in these patients. Methods We collected retrospective data on urogenital system and pubertal development of 86 ADA-SCID patients followed in the period 2000–2017 at the Great Ormond Street Hospital (UK) and 5 centers in Italy. In particular, we recorded clinical history and visits, and routine blood tests and ultrasound scans were performed as part of patients’ follow-up. Results and Discussion We found a higher frequency of congenital and acquired undescended testes compared with healthy children (congenital, 22% in our sample, 0.5–4% described in healthy children; acquired, 16% in our sample, 1–3% in healthy children), mostly requiring orchidopexy. No urogenital abnormalit...
Blood
Karl S Peggs and Sarah J Albon contributed equally to the work and are joint first author Introdu... more Karl S Peggs and Sarah J Albon contributed equally to the work and are joint first author Introduction Alemtuzumab reduces the incidence of GVHD after unrelated donor stem cell transplant (MUD SCT) but delays immune reconstitution resulting in high morbidity/mortality from viral infections. Previous studies have suggested that adoptive transfer of allodepleted donor T cells (ADTs) improves immunity after SCT but this has never been tested in a randomised study. We developed a methodology for selective immunomagnetic depletion of alloreactive T-cells upregulating CD25 and CD71 after activation with host dendritic cells (DC) and showed that ADTs retain anti-viral responses with minimal host alloreactivity (Samarasinghe et al Blood 2010). We have now tested whether ADTs can safely be used to improve immune reconstitution after MUD SCT for haematological malignancies in a randomised Phase II multi-centre clinical study; ICAT (NCT01827579). Methods Patients undergoing Alemtuzumab-based p...
Blood
Chimeric antigen receptor (CAR)19 T-cells exhibit powerful anti-leukemic effects in patients with... more Chimeric antigen receptor (CAR)19 T-cells exhibit powerful anti-leukemic effects in patients with B cell malignancies. However, the complexity of production of patient bespoke T cell products is a major barrier to the broader application of this approach. We are investigating a novel strategy to enable "off-the-shelf"' therapy with mismatched donor CAR19 T cells. Transcription activator-like effector nucleases (TALEN)s can be used to overcome HLA barriers by eliminating the risk of graft-versus-host disease (GvHD) through disruption of T cell receptor expression, and by simultaneously targeting CD52, cells can be rendered insensitive to the lymphodepleting agent Alemtuzumab. Administration of Alemtuzumab can then be exploited to prevent host-mediated rejection of HLA mismatched CAR19 T cells. We manufactured a bank of such cells from volunteer donor T cells under GMP conditions on behalf of Cellectis S.A for final stage validation studies using a third generation self ...
Pediatric Blood & Cancer
The molecular detection of minimal residual disease (MRD) is standard of care in acute lymphoblas... more The molecular detection of minimal residual disease (MRD) is standard of care in acute lymphoblastic leukemia to personalize the stratification of patients to appropriate intensity chemotherapy regimens. High‐throughput sequencing (HTS) techniques are driving changes to MRD methodologies. Our study demonstrates HTS can identify suitable diagnostic markers, even in cases where traditional screening has been unsuccessful. Markers identified by HTS were used to track MRD using standard real‐time quantitative PCR. We show, with six patient examples, clinical benefits of utilizing HTS to screen diagnostic samples and its necessity when traditional screening techniques fail. This is practical evidence that current MRD diagnostic marker screening should be replaced by an HTS approach.
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](Frontiers in immunology, 2018
Juvenile dermatomyositis (JDM) is a rare form of childhood autoimmune myositis that presents with... more Juvenile dermatomyositis (JDM) is a rare form of childhood autoimmune myositis that presents with proximal muscle weakness and skin rash. B cells are strongly implicated in the pathogenesis of the disease, but the underlying mechanisms are unknown. Therefore, the main objective of our study was to investigate mechanisms driving B cell lymphocytosis and define pathological features of B cells in JDM patients. Patients were recruited through the UK JDM Cohort and Biomarker study. Peripheral blood B cell subpopulations were immunophenotyped by flow cytometry. The results identified that immature transitional B cells were significantly expanded in active JDM, actively dividing, and correlated positively with disease activity. Protein and RNAseq analysis revealed high interferon alpha (IFNα) and TLR7-pathway signatures pre-treatment. Stimulation of B cells through TLR7/8 promoted both IL-10 and IL-6 production in controls but failed to induce IL-10 in JDM patient cells. Interrogation of ...
The Journal of molecular diagnostics : JMD, 2018
Certain blood components and anticoagulants interfere with the PCR process and subsequent analysi... more Certain blood components and anticoagulants interfere with the PCR process and subsequent analysis. Here we demonstrate that reliable test results can be obtained for chimerism analysis despite omitting a DNA-extraction step and performing PCR and fragment analysis directly on bone marrow, whole blood, and individual cell fractions. For chimerism analysis, direct-tissue PCR is possible with the use of a robust, commercially available PCR mix containing a DNA polymerase capable of DNA amplification directly from the sample without the need for pretreatment. A total of 178 chimerism samples were processed directly, and results were compared to those obtained from the corresponding DNA sample. No differences were observed between the two sets of results. For the cell fraction-purity assessment, commercially available PCR kits were used directly on T and B cells without the use of any additional lysing agent. A total of 53 purity samples and their corresponding DNA samples were analyzed...
Haematologica, Jan 7, 2017
The Journal of allergy and clinical immunology, Jan 8, 2017
Thymus transplantation is a promising strategy for the treatment of athymic complete DiGeorge syn... more Thymus transplantation is a promising strategy for the treatment of athymic complete DiGeorge syndrome (cDGS). Twelve patients with cDGS were transplanted with allogeneic cultured thymus. To confirm and extend the results previously obtained in a single centre. Two patients died of pre-existing viral infections without developing thymopoeisis and one late death occurred from autoimmune thrombocytopaenia. One infant suffered septic shock shortly after transplant resulting in graft loss and the need for a second transplant. Evidence of thymopoeisis developed from 5-6 months after transplantation in ten patients. The median (range) of circulating naïve CD4 counts (x10(6)/L) were 44(11-440) and 200(5-310) at twelve and twenty-four months post-transplant and T-cell receptor excision circles were 2238 (320-8807) and 4184 (1582 -24596) per10(6) T-cells. Counts did not usually reach normal levels for age but patients were able to clear pre-existing and later-acquired infections. At a median...
Science translational medicine, Jan 25, 2017
Autologous T cells engineered to express chimeric antigen receptor against the B cell antigen CD1... more Autologous T cells engineered to express chimeric antigen receptor against the B cell antigen CD19 (CAR19) are achieving marked leukemic remissions in early-phase trials but can be difficult to manufacture, especially in infants or heavily treated patients. We generated universal CAR19 (UCART19) T cells by lentiviral transduction of non-human leukocyte antigen-matched donor cells and simultaneous transcription activator-like effector nuclease (TALEN)-mediated gene editing of T cell receptor α chain and CD52 gene loci. Two infants with relapsed refractory CD19(+) B cell acute lymphoblastic leukemia received lymphodepleting chemotherapy and anti-CD52 serotherapy, followed by a single-dose infusion of UCART19 cells. Molecular remissions were achieved within 28 days in both infants, and UCART19 cells persisted until conditioning ahead of successful allogeneic stem cell transplantation. This bridge-to-transplantation strategy demonstrates the therapeutic potential of gene-editing technol...
Journal of Allergy and Clinical Immunology, 2016
Background: Signalling through the T cell receptor (TCR) is critical for T cell development and f... more Background: Signalling through the T cell receptor (TCR) is critical for T cell development and function. Linker for activation of T cells (LAT) is a transmembrane adaptor signalling molecule that is part of the TCR complex and is essential for T cell development as demonstrated by LAT-deficient mice which show a complete lack of peripheral T cells. Objective: We describe a pedigree affected by a severe combined immunodeficiency (SCID) phenotype with absent T cells and normal B cells and natural killer (NK) cells. A novel homozygous frameshift mutation in the gene encoding for LAT was identified in this kindred. Methods: Genetic, molecular and functional analyses were used to identify and characterise the LAT defect. Clinical and immunological analysis of patients was also performed and reported. Results: Homozygosity mapping was used to identify potential defective genes. Sanger sequencing of the LAT gene showed a mutation that resulted in a premature stop codon and protein truncation leading to complete loss-of-function and loss-of-expression of LAT in the affected family members. We also demonstrate loss of LAT expression and lack of TCR signalling restoration in LAT-deficient cell lines reconstituted with a synthetic LAT gene bearing this SCID mutation. Conclusion: The results of this study shows for the first time that inherited LAT deficiency should be considered in patients with combined immunodeficiency with T cell abnormalities.
The Journal of allergy and clinical immunology, Oct 20, 2016
Reduced-intensity conditioning (RIC) regimens are increasingly being used in the transplantation ... more Reduced-intensity conditioning (RIC) regimens are increasingly being used in the transplantation of patients with primary immunodeficiency disorders (PIDs), but there are no large studies looking at long-term lineage-specific chimerism. We sought to analyze long-term chimerism and event-free survival in children undergoing transplantation for PIDs using RIC with fludarabine and melphalan (Flu/Melph) and to study the effect of donor type and stem cell source. One hundred forty-two children underwent transplantation with RIC by using Flu/Melph and for PIDs by using bone marrow (n = 93) or peripheral blood stem cells (PBSCs; n = 49). Donors were matched unrelated donors (n = 72), mismatched unrelated donors (n = 37), matched sibling donors (n = 14), matched family donors (n = 12), and mismatched family donors (n = 7). Overall survival at a median follow-up of 7.5 years was 78%, irrespective of stem cell source or donor type. When bone marrow was used as the stem cell source, 26% of pat...
Journal of Clinical Immunology, 2016
Pediatric Transplantation, 2020
Paediatric heart transplantation recipients suffer an increased incidence of infectious, autoimmu... more Paediatric heart transplantation recipients suffer an increased incidence of infectious, autoimmune and allergic problems. The relative roles of thymus excision and immunosuppressive treatments in contributing to these sequelae are not clear. We compared the immunological phenotypes of 25 heart transplant recipients (Tx), 10 children who underwent thymus excision during non‐transplantation cardiac surgery (TE) and 25 age range–matched controls, in two age bands: 1‐9 and 10‐16 years. Significant differences from controls were seen mainly in the younger age band with Tx showing lower CD3 and CD4 cell counts whilst TE showed lower CD8 cell counts. Naïve T cell and recent thymic emigrant proportions and counts were significantly lower than controls in both groups in the lower age band. T cell recombination excision circle (TREC) levels were lower than controls in both groups in both age bands. There were no differences in regulatory T cells, but in those undergoing thymus excision in infancy, their proportions were higher in TE than Tx, a possible direct effect of immunosuppression. T cell receptor V beta spectratyping showed fewer peaks in both groups than in controls (predominantly in the older age band). Thymus excision in infancy was associated with lower CD8 cell counts and higher proportions of Tregs in TE compared to Tx. These data are consistent with thymus excision, particularly in infancy, being the most important influence on immunological phenotype after heart transplantation.
Blood
Historically, survival and activity of individual human hematopoietic progenitor subtypes have be... more Historically, survival and activity of individual human hematopoietic progenitor subtypes have been studied exclusively via transplantation in permissive mouse models. Despite being a relevant investigational tool, such animal models do not recapitulate the human hematopoietic milieu. Specifically, the production of human T and NK cells in these mice is severely impaired therefore, as of today, it has been impossible to measure with enough confidence the in vivo dynamics of human T/NK lymphoid progenitors. Hematopoietic stem cell gene therapy (GT) using integrating viral vectors has opened a unique opportunity to trace the fate of transplanted cells for the first time directly in vivo in humans by means of integration sites (IS) clonal tracking. Our mathematical modelling of IS data in clinical GT supported the existence of a population of long-term lymphoid progenitors (LtLP) capable of surviving independently from hematopoietic stem cells (HSC). However, to date, no experimental s...
Frontiers in Immunology
It is intriguing that, unlike adults with HIV-1, children with HIV-1 reach a greater CD4+ T cell ... more It is intriguing that, unlike adults with HIV-1, children with HIV-1 reach a greater CD4+ T cell recovery following planned treatment cessation. The reasons for the better outcomes in children remain unknown but may be related to increased thymic output and diversity of T cell receptor repertoires. HIV-1 infected children from the PENTA 11 trial tolerated planned treatment interruption without adverse long-term clinical, virological, or immunological consequences, once antiretroviral therapy was re-introduced. This contrasts to treatment interruption trials of HIV-1 infected adults, who had rapid changes in T cells and slow recovery when antiretroviral therapy was restarted. How children can develop such effective immune responses to planned treatment interruption may be critical for future studies. PENTA 11 was a randomized, phase II trial of planned treatment interruptions in HIV-1-infected children (ISRCTN 36694210). In this sub-study, eight patients in long-term follow-up were c...
Science Translational Medicine, 2011
X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the common cytokine... more X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the common cytokine receptor g chain. These mutations classically lead to complete absence of functional T and natural killer cell lineages as well as to intrinsically compromised B cell function. Although human leukocyte antigen (HLA)-matched hematopoietic stem cell transplantation (HSCT) is highly successful in SCID-X1 patients, HLA-mismatched procedures can be associated with prolonged immunodeficiency, graft-versus-host disease, and increased overall mortality. Here, 10 children were treated with autologous CD34+ hematopoietic stem and progenitor cells transduced with a conventional gammaretroviral vector. The patients did not receive myelosuppressive conditioning and were monitored for immunological recovery after cell infusion. All patients were alive after a median follow-up of 80 months (range, 54 to 107 months), and a functional polyclonal T cell repertoire was restored in all patients. Humoral im...
Blood Advances
The action of hematopoietic cell transplantation in controlling leukemia is principally mediated ... more The action of hematopoietic cell transplantation in controlling leukemia is principally mediated by donor T cells directed against residual recipient malignant cells. However, its utility is limited by graft-versus-host disease (GVHD), where alloreactivity is extended beyond leukemic and marrow cells. In a human/murine chimeric model, we previously showed that the preferential infiltration of cord blood (CB) CD8+ T cells eradicates an Epstein-Barr virus–driven lymphoblastoid tumor without causing xenogeneic GVHD. In the clinic, however, cord blood CD8+ T-cell reconstitution is significantly delayed, and the observation of such a robust antileukemia effect mediated by cord blood CD8+ T cells has not been reported. We describe an observation of very early T-cell expansion in 4 high-risk pediatric leukemia patients receiving third-party, pooled granulocytes after T cell–replete CB transplantation (CBT). The T-cell expansion was transient but robust, including expansion of CD8+ T cells,...
International Journal of Neonatal Screening
Spinal muscular atrophy (SMA) is an autosomal inherited neuromuscular genetic disease caused, in ... more Spinal muscular atrophy (SMA) is an autosomal inherited neuromuscular genetic disease caused, in 95% of cases, by homozygous deletions involving the SMN1 gene exon 7. It remains the leading cause of death in children under 2 years of age. New treatments have been developed and adopted for use in many countries, including the UK. Success of these treatments depends on early diagnosis and intervention in newborn babies, and many countries have implemented a newborn screening (NBS) or pilot NBS program to detect SMN1 exon 7 deletions on dried blood spots. In the UK, there is no current NBS program for SMA, and no pilot studies have commenced. For consideration of adoption of NBS for a new condition, numerous criteria must be satisfied, including critical assessment of a working methodology. This study uses a commercially available real-time PCR assay to simultaneously detect two different DNA segments (SMN1 exon 7 and control gene RPP30) using DNA extracted from a dried blood spot. Thi...
Journal of Clinical Immunology
Background Improved survival in ADA-SCID patients is revealing new aspects of the systemic disord... more Background Improved survival in ADA-SCID patients is revealing new aspects of the systemic disorder. Although increasing numbers of reports describe the systemic manifestations of adenosine deaminase deficiency, currently there are no studies in the literature evaluating genital development and pubertal progress in these patients. Methods We collected retrospective data on urogenital system and pubertal development of 86 ADA-SCID patients followed in the period 2000–2017 at the Great Ormond Street Hospital (UK) and 5 centers in Italy. In particular, we recorded clinical history and visits, and routine blood tests and ultrasound scans were performed as part of patients’ follow-up. Results and Discussion We found a higher frequency of congenital and acquired undescended testes compared with healthy children (congenital, 22% in our sample, 0.5–4% described in healthy children; acquired, 16% in our sample, 1–3% in healthy children), mostly requiring orchidopexy. No urogenital abnormalit...
Blood
Karl S Peggs and Sarah J Albon contributed equally to the work and are joint first author Introdu... more Karl S Peggs and Sarah J Albon contributed equally to the work and are joint first author Introduction Alemtuzumab reduces the incidence of GVHD after unrelated donor stem cell transplant (MUD SCT) but delays immune reconstitution resulting in high morbidity/mortality from viral infections. Previous studies have suggested that adoptive transfer of allodepleted donor T cells (ADTs) improves immunity after SCT but this has never been tested in a randomised study. We developed a methodology for selective immunomagnetic depletion of alloreactive T-cells upregulating CD25 and CD71 after activation with host dendritic cells (DC) and showed that ADTs retain anti-viral responses with minimal host alloreactivity (Samarasinghe et al Blood 2010). We have now tested whether ADTs can safely be used to improve immune reconstitution after MUD SCT for haematological malignancies in a randomised Phase II multi-centre clinical study; ICAT (NCT01827579). Methods Patients undergoing Alemtuzumab-based p...
Blood
Chimeric antigen receptor (CAR)19 T-cells exhibit powerful anti-leukemic effects in patients with... more Chimeric antigen receptor (CAR)19 T-cells exhibit powerful anti-leukemic effects in patients with B cell malignancies. However, the complexity of production of patient bespoke T cell products is a major barrier to the broader application of this approach. We are investigating a novel strategy to enable "off-the-shelf"' therapy with mismatched donor CAR19 T cells. Transcription activator-like effector nucleases (TALEN)s can be used to overcome HLA barriers by eliminating the risk of graft-versus-host disease (GvHD) through disruption of T cell receptor expression, and by simultaneously targeting CD52, cells can be rendered insensitive to the lymphodepleting agent Alemtuzumab. Administration of Alemtuzumab can then be exploited to prevent host-mediated rejection of HLA mismatched CAR19 T cells. We manufactured a bank of such cells from volunteer donor T cells under GMP conditions on behalf of Cellectis S.A for final stage validation studies using a third generation self ...
Pediatric Blood & Cancer
The molecular detection of minimal residual disease (MRD) is standard of care in acute lymphoblas... more The molecular detection of minimal residual disease (MRD) is standard of care in acute lymphoblastic leukemia to personalize the stratification of patients to appropriate intensity chemotherapy regimens. High‐throughput sequencing (HTS) techniques are driving changes to MRD methodologies. Our study demonstrates HTS can identify suitable diagnostic markers, even in cases where traditional screening has been unsuccessful. Markers identified by HTS were used to track MRD using standard real‐time quantitative PCR. We show, with six patient examples, clinical benefits of utilizing HTS to screen diagnostic samples and its necessity when traditional screening techniques fail. This is practical evidence that current MRD diagnostic marker screening should be replaced by an HTS approach.
Frontiers in immunology, 2018
Juvenile dermatomyositis (JDM) is a rare form of childhood autoimmune myositis that presents with... more Juvenile dermatomyositis (JDM) is a rare form of childhood autoimmune myositis that presents with proximal muscle weakness and skin rash. B cells are strongly implicated in the pathogenesis of the disease, but the underlying mechanisms are unknown. Therefore, the main objective of our study was to investigate mechanisms driving B cell lymphocytosis and define pathological features of B cells in JDM patients. Patients were recruited through the UK JDM Cohort and Biomarker study. Peripheral blood B cell subpopulations were immunophenotyped by flow cytometry. The results identified that immature transitional B cells were significantly expanded in active JDM, actively dividing, and correlated positively with disease activity. Protein and RNAseq analysis revealed high interferon alpha (IFNα) and TLR7-pathway signatures pre-treatment. Stimulation of B cells through TLR7/8 promoted both IL-10 and IL-6 production in controls but failed to induce IL-10 in JDM patient cells. Interrogation of ...
The Journal of molecular diagnostics : JMD, 2018
Certain blood components and anticoagulants interfere with the PCR process and subsequent analysi... more Certain blood components and anticoagulants interfere with the PCR process and subsequent analysis. Here we demonstrate that reliable test results can be obtained for chimerism analysis despite omitting a DNA-extraction step and performing PCR and fragment analysis directly on bone marrow, whole blood, and individual cell fractions. For chimerism analysis, direct-tissue PCR is possible with the use of a robust, commercially available PCR mix containing a DNA polymerase capable of DNA amplification directly from the sample without the need for pretreatment. A total of 178 chimerism samples were processed directly, and results were compared to those obtained from the corresponding DNA sample. No differences were observed between the two sets of results. For the cell fraction-purity assessment, commercially available PCR kits were used directly on T and B cells without the use of any additional lysing agent. A total of 53 purity samples and their corresponding DNA samples were analyzed...
Haematologica, Jan 7, 2017
The Journal of allergy and clinical immunology, Jan 8, 2017
Thymus transplantation is a promising strategy for the treatment of athymic complete DiGeorge syn... more Thymus transplantation is a promising strategy for the treatment of athymic complete DiGeorge syndrome (cDGS). Twelve patients with cDGS were transplanted with allogeneic cultured thymus. To confirm and extend the results previously obtained in a single centre. Two patients died of pre-existing viral infections without developing thymopoeisis and one late death occurred from autoimmune thrombocytopaenia. One infant suffered septic shock shortly after transplant resulting in graft loss and the need for a second transplant. Evidence of thymopoeisis developed from 5-6 months after transplantation in ten patients. The median (range) of circulating naïve CD4 counts (x10(6)/L) were 44(11-440) and 200(5-310) at twelve and twenty-four months post-transplant and T-cell receptor excision circles were 2238 (320-8807) and 4184 (1582 -24596) per10(6) T-cells. Counts did not usually reach normal levels for age but patients were able to clear pre-existing and later-acquired infections. At a median...
Science translational medicine, Jan 25, 2017
Autologous T cells engineered to express chimeric antigen receptor against the B cell antigen CD1... more Autologous T cells engineered to express chimeric antigen receptor against the B cell antigen CD19 (CAR19) are achieving marked leukemic remissions in early-phase trials but can be difficult to manufacture, especially in infants or heavily treated patients. We generated universal CAR19 (UCART19) T cells by lentiviral transduction of non-human leukocyte antigen-matched donor cells and simultaneous transcription activator-like effector nuclease (TALEN)-mediated gene editing of T cell receptor α chain and CD52 gene loci. Two infants with relapsed refractory CD19(+) B cell acute lymphoblastic leukemia received lymphodepleting chemotherapy and anti-CD52 serotherapy, followed by a single-dose infusion of UCART19 cells. Molecular remissions were achieved within 28 days in both infants, and UCART19 cells persisted until conditioning ahead of successful allogeneic stem cell transplantation. This bridge-to-transplantation strategy demonstrates the therapeutic potential of gene-editing technol...
Journal of Allergy and Clinical Immunology, 2016
Background: Signalling through the T cell receptor (TCR) is critical for T cell development and f... more Background: Signalling through the T cell receptor (TCR) is critical for T cell development and function. Linker for activation of T cells (LAT) is a transmembrane adaptor signalling molecule that is part of the TCR complex and is essential for T cell development as demonstrated by LAT-deficient mice which show a complete lack of peripheral T cells. Objective: We describe a pedigree affected by a severe combined immunodeficiency (SCID) phenotype with absent T cells and normal B cells and natural killer (NK) cells. A novel homozygous frameshift mutation in the gene encoding for LAT was identified in this kindred. Methods: Genetic, molecular and functional analyses were used to identify and characterise the LAT defect. Clinical and immunological analysis of patients was also performed and reported. Results: Homozygosity mapping was used to identify potential defective genes. Sanger sequencing of the LAT gene showed a mutation that resulted in a premature stop codon and protein truncation leading to complete loss-of-function and loss-of-expression of LAT in the affected family members. We also demonstrate loss of LAT expression and lack of TCR signalling restoration in LAT-deficient cell lines reconstituted with a synthetic LAT gene bearing this SCID mutation. Conclusion: The results of this study shows for the first time that inherited LAT deficiency should be considered in patients with combined immunodeficiency with T cell abnormalities.
The Journal of allergy and clinical immunology, Oct 20, 2016
Reduced-intensity conditioning (RIC) regimens are increasingly being used in the transplantation ... more Reduced-intensity conditioning (RIC) regimens are increasingly being used in the transplantation of patients with primary immunodeficiency disorders (PIDs), but there are no large studies looking at long-term lineage-specific chimerism. We sought to analyze long-term chimerism and event-free survival in children undergoing transplantation for PIDs using RIC with fludarabine and melphalan (Flu/Melph) and to study the effect of donor type and stem cell source. One hundred forty-two children underwent transplantation with RIC by using Flu/Melph and for PIDs by using bone marrow (n = 93) or peripheral blood stem cells (PBSCs; n = 49). Donors were matched unrelated donors (n = 72), mismatched unrelated donors (n = 37), matched sibling donors (n = 14), matched family donors (n = 12), and mismatched family donors (n = 7). Overall survival at a median follow-up of 7.5 years was 78%, irrespective of stem cell source or donor type. When bone marrow was used as the stem cell source, 26% of pat...
Journal of Clinical Immunology, 2016