Peter J. Shaw

Expansion of Functionally Immature Transitional B Cells Is Associated with Human-Immunodeficient States Characterized by Impaired Humoral Immunity

X-linked lymphoproliferative disease (XLP) is a severe immunodeficiency associated with a marked reduction in circulating memory B cells. Our investigation of the B cell compartment of XLP patients revealed an increase in the frequency of a population of B cells distinct from those previously defined. This population displayed increased expression of CD10, CD24, and CD38, indicating that it could consist of circulating immature/transitional B cells. Supporting this possibility, CD10+CD24highCD38high B cells displayed other immature characteristics, including unmutated Ig V genes and elevated levels of surface IgM; they also lacked expression of Bcl-2 and a panel of activation molecules. The capacity of CD24highCD38high B cells to proliferate, secrete Ig, and migrate in vitro was greatly reduced compared with mature B cell populations. Moreover, CD24highCD38high B cells were increased in the peripheral blood of neonates, patients with common variable immunodeficiency, and patients recovering from hemopoietic stem cell transplant. Thus, an expansion of functionally immature B cells may contribute to the humoral immunodeficient state that is characteristic of neonates, as well as patients with XLP or common variable immunodeficiency, and those recovering from a stem cell transplant. Further investigation of transitional B cells will improve our understanding of human B cell development and how alterations to this process may precipitate immunodeficiency or autoimmunity.

Reduced-intensity conditioning and HLA-matched haemopoietic stem-cell transplantation in patients with chronic granulomatous disease: a prospective multicentre study

BACKGROUND In chronic granulomatous disease allogeneic haemopoietic stem-cell transplantation (HSCT) in adolescents and young adults and patients with high-risk disease is complicated by graft-failure, graft-versus-host disease (GVHD), and transplant-related mortality. We examined the effect of a reduced-intensity conditioning regimen designed to enhance myeloid engraftment and reduce organ toxicity in these patients. METHODS This prospective study was done at 16 centres in ten countries worldwide. Patients aged 0-40 years with chronic granulomatous disease were assessed and enrolled at the discretion of individual centres. Reduced-intensity conditioning consisted of high-dose fludarabine (30 mg/m(2) [infants <9 kg 1·2 mg/kg]; one dose per day on days -8 to -3), serotherapy (anti-thymocyte globulin [10 mg/kg, one dose per day on days -4 to -1; or thymoglobuline 2·5 mg/kg, one dose per day on days -5 to -3]; or low-dose alemtuzumab [<1 mg/kg on days -8 to -6]), and low-dose (50-72% of myeloablative dose) or targeted busulfan administration (recommended cumulative area under the curve: 45-65 mg/L × h). Busulfan was administered mainly intravenously and exceptionally orally from days -5 to -3. Intravenous busulfan was dosed according to weight-based recommendations and was administered in most centres (ten) twice daily over 4 h. Unmanipulated bone marrow or peripheral blood stem cells from HLA-matched related-donors or HLA-9/10 or HLA-10/10 matched unrelated-donors were infused. The primary endpoints were overall survival and event-free survival (EFS), probabilities of overall survival and EFS at 2 years, incidence of acute and chronic GVHD, achievement of at least 90% myeloid donor chimerism, and incidence of graft failure after at least 6 months of follow-up. FINDINGS 56 patients (median age 12·7 years; IQR 6·8-17·3) with chronic granulomatous disease were enrolled from June 15, 2003, to Dec 15, 2012. 42 patients (75%) had high-risk features (ie, intractable infections and autoinflammation), 25 (45%) were adolescents and young adults (age 14-39 years). 21 HLA-matched related-donor and 35 HLA-matched unrelated-donor transplants were done. Median time to engraftment was 19 days (IQR 16-22) for neutrophils and 21 days (IQR 16-25) for platelets. At median follow-up of 21 months (IQR 13-35) overall survival was 93% (52 of 56) and EFS was 89% (50 of 56). The 2-year probability of overall survival was 96% (95% CI 86·46-99·09) and of EFS was 91% (79·78-96·17). Graft-failure occurred in 5% (three of 56) of patients. The cumulative incidence of acute GVHD of grade III-IV was 4% (two of 56) and of chronic graft-versus-host disease was 7% (four of 56). Stable (≥90%) myeloid donor chimerism was documented in 52 (93%) surviving patients. INTERPRETATION This reduced-intensity conditioning regimen is safe and efficacious in high-risk patients with chronic granulomatous disease. FUNDING None.

Chimerism and Tolerance in a Recipient of a Deceased-Donor Liver Transplant

Complete hematopoietic chimerism and tolerance of a liver allograft from a deceased male donor developed in a 9-year-old girl, with no evidence of graft-versus-host disease 17 months after transplantation. The tolerance was preceded by a period of severe hemolysis, reflecting partial chimerism that was refractory to standard therapies. The hemolysis resolved after the gradual withdrawal of all immunosuppressive therapy.

Early and late invasive pneumococcal infection following stem cell transplantation: a European Bone Marrow Transplantation survey

Summary.  Streptococcus pneumoniae (S. pneumoniae) may cause severe and lethal infections months and years following stem cell transplantation (SCT). In a prospective survey over a 3·5‐year period, we assessed the incidence, risk factors and outcome for invasive pneumococcal infection (IPI) following SCT. Fifty‐one episodes of IPI were reported: 43 episodes after bone marrow transplantation (BMT) and 8 after peripheral blood stem cell transplantation (PBSCT); 35 after allogeneic SCT and 16 after autologous SCT. Seven IPI episodes, all bacteraemias, were defined as early, occurring 1–35 d (median 3 d) post transplantation. Forty‐four episodes were defined as late (≥ 100 d post SCT), occurring 4 months to 10 years (median 17 months) post transplantation. The incidences of early and late IPI were 2·03/1000 and 8·63/1000 transplantations respectively (P = 0·001). A higher incidence of late IPI was observed after BMT than after PBSCT (10·99 versus 3·23/1000; P < 0·01) and after allogeneic versus autologous SCT (12·20 versus 4·60/1000; P < 0·01). There was a higher estimated incidence of IPI in allogeneic patients with than in those without graft‐versus‐host disease (GVHD) (18·85 versus 8·25/1000; P = 0·015). The mortality rate was 20%, including 2/7 of early and 8/44 of late IPI. S. pneumoniae is a rare but important complication during the aplastic phase after SCT. In conclusion, S. pneumoniae is a significant cause of morbidity late post‐transplantation, especially in allogeneic patients, and particularly those with GVHD. The high IPI mortality rate, both early and late post‐transplantation, requires preventive approaches, mainly effective immunization.

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

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.

European guidelines for diagnosis and treatment of adenovirus infection in leukemia and stem cell transplantation: summary of ECIL‐4 (2011)

Human adenovirus (HAdV) infections are increasingly recognized as important pathogens in immunocompromised hosts, especially in patients with severely suppressed T‐cell function. The 4th European Conference of Infections in Leukemia (ECIL‐4) has developed evidence‐based guidelines for diagnosis and management of HAdV infections. The risk for HAdV‐associated disease is increased in children, and risk factors for HAdV disease are T‐cell depletion, unrelated and cord blood hematopoietic stem cell transplantation, graft‐versus‐host disease grades III–IV, and lymphopenia. The recommended technique for monitoring of high‐risk patients is quantitative polymerase chain reaction. Cidofovir is the most used antiviral therapy, although no controlled study has been performed. HAdV‐specific T‐cell therapy is in development.

Plasma Epstein-Barr Virus (EBV) DNA Is a Biomarker for EBV-Positive Hodgkin's Lymphoma

Purpose: Latent Epstein-Barr virus (EBV) genomes are found in the malignant cells of approximately one-third of Hodgkins lymphoma (HL) cases. Detection and quantitation of EBV viral DNA could potentially be used as a biomarker of disease activity. Experimental Design: Initially, EBV-DNA viral load was prospectively monitored from peripheral blood mononuclear cells (PBMC) in patients with HL. Subsequently, we analyzed viral load in plasma from a second cohort of patients. A total of 58 patients with HL (31 newly diagnosed, 6 relapsed, and 21 in long-term remission) were tested. Using real-time PCR, 43 PBMC and 52 plasma samples were analyzed. Results: EBV-DNA was detectable in the plasma of all EBV-positive patients with HL prior to therapy. However, viral DNA was undetectable following therapy in responding patients (P = 0.0156), EBV-positive HL patients in long-term remission (P = 0.0011), and in all patients with EBV-negative HL (P = 0.0238). Conversely, there was no association seen for the EBV-DNA load measured from PBMC in patients with active EBV-positive HL patients as compared with EBV-negative HL, or patients in long-term remission. EBV-DNA load in matched plasma/PBMC samples were not correlated. Conclusions: We show that free plasma EBV-DNA has excellent sensitivity and specificity, and can be used as a noninvasive biomarker for EBV-positive HL and that serial monitoring could predict response to therapy. Additional prospective studies are required to further evaluate the use of free plasma EBV-DNA as a biomarker for monitoring response to treatment in patients with EBV-positive HL.

Improved outcomes of children with malignancy admitted to a pediatric intensive care unit.

ObjectiveTo assess the acute and long-term outcomes of children admitted to the intensive care unit with cancer or complications after bone marrow transplantation. DesignRetrospective analysis of databases from a prospective pediatric intensive care unit (PICU) database supplemented by case notes review. SettingA PICU in a tertiary pediatric hospital. PatientsAll children with malignancy admitted to the PICU between May 1, 1987, and April 30, 1996. InterventionsNone. Measurements and Main ResultsThere were 206 admissions to the PICU during a 9-yr study period of 150 children with malignancies or complications after bone marrow transplantation. Forty patients died in the PICU (27% mortality rate). The most frequent indications for PICU admission were shock and respiratory disease. Of 56 children admitted with shock, there were 16 deaths (29% mortality rate). In 24 episodes of sepsis, inotropic and ventilatory support were required and 13 patients (54%) survived. Analysis of long-term survival gave estimates of 50% survival for all oncology patients admitted to the PICU and 42% for those admitted for shock. ConclusionsA high proportion of oncology patients admitted to the PICU requiring intensive intervention survive and go on to be cured of their malignancy. Our study suggests the PICU outcome for these patients has improved.

Role of prospective screening of blood for invasive aspergillosis by polymerase chain reaction in febrile neutropenic recipients of haematopoietic stem cell transplants and patients with acute leukaemia.

Guidelines for the use of polymerase chain reaction (PCR)‐based assays to aid the diagnosis of invasive aspergillosis (IA) in high‐risk haematology patients have not been formulated. We prospectively evaluated a nested PCR assay to detect Aspergillus in blood during 95 febrile neutropenic episodes, in patients with haematological malignancy and haematopoietic stem cell transplant (HSCT) recipients. PCR results were correlated with the diagnostic classification of the 2002 European Organisation for Research and Treatment of Cancer/Mycosis Study Group. When two‐positive results were used to define an episode as ‘PCR positive’, the sensitivity, specificity, positive‐predictive value and negative predictive value for ‘proven’/‘probable’ IA (n = 13) were 100%, 75·4%, 46·4% and 100%, respectively. Consecutive positive results occurred in 61·5% of these 13 episodes. Overall, PCR positivity preceded standard diagnosis by a mean of 14 d and the median time between positive results was shorter than that in other categories of IA. All 13 episodes occurred in the setting of allogeneic HSCT recipients and acute leukaemia. If ‘eligibility’ for antifungal therapy were based on two‐positive‐PCR tests, use of empiric treatment could have been reduced by up to 37%. The nested PCR assay is a practical screening test for excluding IA. Patients with consecutive positive results or intermittent‐positive results (within 14 d) warrant immediate investigations for IA and the initiation of antifungal therapy.

Differential expression of CD21 identifies developmentally and functionally distinct subsets of human transitional B cells

The transitional stage of B-cell development represents an important step where autoreactive cells are deleted, allowing the generation of a mature functional B-cell repertoire. In mice, 3 subsets of transitional B cells have been identified. In contrast, most studies of human transitional B cells have focused on a single subset defined as CD24(hi)CD38(hi) B cells. Here, we have identified 2 subsets of human transitional B cells based on the differential expression of CD21. CD21(hi) transitional cells displayed higher expression of CD23, CD44, and IgD, and exhibited greater proliferation and Ig secretion in vitro than CD21(lo) transitional B cells. In contrast, the CD21(lo) subset expressed elevated levels of LEF1, a transcription factor highly expressed by immature lymphocytes, and produced higher amounts of autoreactive Ab. These phenotypic, functional, and molecular features suggest that CD21(lo) transitional B cells are less mature than the CD21(hi) subset. This was confirmed by analyzing X-linked agammaglobulinemia patients and the kinetics of B-cell reconstitution after stem cell transplantation, which revealed that the development of CD21(lo) transitional B cells preceded that of CD21(hi) transitional cells. These findings provide important insights into the process of human B-cell development and have implications for understanding the processes underlying perturbed B-cell maturation in autoimmune and immunodeficient conditions.