Persistence of HIV reservoir following successful haematopoietic stem cell transplant for juvenile myelomonocytic leukaemia in a child with perinatally acquired HIV.

This report describes a case of juvenile myelomonocytic leukaemia (JMML) on a background of both perinatally acquired HIV infection and congenital cytomegalovirus, and management of antiretroviral therapy during haematopoietic stem cell transplant. Peripheral blood HIV viral load remained below the lower limit of detection throughout and following transplant and is currently <20 RNA copies/mL. The child is currently in remission from JMML, but HIV DNA remains detectable despite myeloablative conditioning and sustained plasma HIV viral suppression.

Alternative donor SCT for the treatment of MHC class II deficiency.

MHC Class II deficiency is a rare primary immunodeficiency disease characterized by absent HLA Class II expression resulting in CD4 lymphopenia, lack of Ag-specific responses and recurrent infection. Without successful allogeneic SCT, most children succumb to infection within the first decade of life. To date, alternative donor transplants for this disorder have been inferior to SCT for other forms of combined immunodeficiency disease due to an increased incidence of graft rejection, GVHD and death from infections generally acquired before haematopoietic cell transplantation. This study details the transplant outcome of 16 affected children consecutively transplanted at four centers since 1990, 8 of whom required mechanical ventilation pretransplant. Stem cells were derived from an HLA-mismatched family member (n=10), an HLA-matched unrelated adult donor (n=4), or an unrelated cord blood donor (n=2). Graft failure occurred in five children, all of whom underwent a second SCT. Six patients developed acute GVHD although no patient developed chronic GVHD after primary transplantation. CD4 T-cell reconstitution remained below the normal range for age, suggesting defective thymopoiesis after allo-SCT. Nonetheless, 69% of children survive without GVHD at a median follow-up of 5.7 years, indicating improved outcomes compared with previous studies.

Impact of viral reactivations in the era of pre-emptive antiviral drug therapy following allogeneic haematopoietic SCT in paediatric recipients.

While pre-emptive rituximab therapy for EBV has substantially reduced the incidence of post-transplant lymphoproliferative disorder, following allogeneic haematopoietic SCT (HSCT), cytomegalovirus (CMV) and adenovirus (ADV) still contribute to significant morbidity and mortality after HSCT. We therefore aimed to identify high-risk children who could benefit from recent advances in virus-specific immunotherapy, define the impact of viral reactivations on survival and estimate the economic burden of pre-emptive antiviral drug therapy. Between 2005 and 2010, prospective monitoring of 291 paediatric HSCT procedures revealed that reactivation of CMV (16%), ADV (15%) and EBV (11%) was frequent during period of CD4 T-cell lymphopenia (0.15 × 10(9) L(-1); P<0.05). We report significant risk factors for reactivation, most notably the use of serotherapy and development of GVHD (grade II) in the presence of pre-existing infection (ADV) or donor and/or recipient seropositivity (CMV, EBV). Most interestingly, CMV and ADV viraemia were the major independent predictors of mortality (P<0.05). CMV, ADV or EBV viral reactivation caused prolonged hospitalization (P<0.05), accounted for 15% of all mortality and substantially increased the cost of transplantation by ∼£22 500 ($34 000). This provides an economic rationale for targeting high-risk HSCT recipients with interventions such as virus-specific cell therapy.

Progress and prospects: gene therapy for inherited immunodeficiencies.

Haematopoietic stem cell transplantation (HSCT) is now widely used to treat primary immunodeficiencies (PID). For patients with specific disorders (severe combined immunodeficiency (SCID)-X1, adenosine deaminase deficiency (ADA)-SCID, X-chronic granulomatous disease (CGD) and Wiskott-Aldrich Syndrome (WAS)) who lack a suitable human leukocyte antigen (HLA)-matched donor, gene therapy has offered an important alternative treatment option. The success of gene therapy can be attributed, in part, to the selective advantage offered to gene-corrected cells, the avoidance of graft-versus-host disease and to the use of pre-conditioning in patients with chemotherapy to facilitate engraftment of corrected cells. Adverse events have been encountered and this has led to detailed characterization of retroviral vector integration profiles. A new generation of self-inactivating retroviral and lentiviral vectors have been designed to address these safety concerns, and are at an advanced stage of preparation for the next phase of clinical testing.

Cell-specific and efficient expression in mouse and human B cells by a novel hybrid immunoglobulin promoter in a lentiviral vector.

The expression of genes specifically in B cells is of great interest in both experimental immunology as well as in future clinical gene therapy. We have constructed a novel enhanced B cell-specific promoter (Igk-E) consisting of an immunoglobulin kappa (Igk) minimal promoter combined with an intronic enhancer sequence and a 3' enhancer sequence from Ig genes. The Igk-E promoter was cloned into a lentiviral vector and used to control expression of enhanced green fluorescent protein (eGFP). Transduction of murine B-cell lymphoma cell lines and activated primary splenic B cells, with IgK-E-eGFP lentivirus, resulted in expression of eGFP, as analysed by flow cytometry, whereas expression in non-B cells was absent. The specificity of the promoter was further examined by transducing Lin(-) bone marrow with Igk-E-eGFP lentivirus and reconstituting lethally irradiated mice. After 16 weeks flow cytometry of lymphoid tissues revealed eGFP expression by CD19+ cells, but not by CD3+, CD11b+, CD11c+ or Gr-1+ cells. CD19+ cells were comprised of both marginal zone B cells and recirculating follicular B cells. Activated human peripheral mononuclear cells were also transduced with Igk-E-eGFP lentivirus under conditions of selective B-cell activation. The Igk-E promoter was able to drive expression of eGFP only in CD19+ cells, while eGFP was expressed by both spleen focus-forming virus and cytomegalovirus constitutive promoters in CD19+ and CD3+ lymphocytes. These data demonstrate that in these conditions the Igk-E promoter is cell specific and controls efficient expression of a reporter protein in mouse and human B cells in the context of a lentiviral vector.

T cell suicide gene therapy to aid haematopoietic stem cell transplantation.

Graft versus host disease (GVHD) is a T cell mediated phenomenon that arises following allogeneic haematopoietic stem cell transplantation, and may be particularly severe in the context of human leukocyte antigen (HLA) mismatched procedures. Although GVHD can be largely abrogated through T cell depletion, such measures result in loss of graft potency and reduced anti-viral and anti-leukaemic effects. The genetic modification of T cells to carry a suicide gene mechanism has been advocated as means of allowing T cells to be harnessed for their beneficial effects, and safely eliminated in the event of significant GVHD. The feasibility of the strategy has been demonstrated in clinical studies using T cells modified by retroviral transduction to encode the herpes simplex thymidine kinase (HSVTK) gene to treat patients with haematological malignancies. However, a number of limitations associated with current protocols have become apparent. Most notably, the process of retroviral transduction, which requires pre-activation of T cells, appears to impair subsequent functional potential. Efforts are now directed towards circumventing the pre-activation requirements of retroviral vectors by using alternative lentiviral systems, in association with improved suicide gene/prodrug combinations.

T cell transduction and suicide with an enhanced mutant thymidine kinase.

Retroviral transfer of Herpes simplex virus thymidine kinase to T cells has been used to confer sensitivity to the antiviral agent ganciclovir. This has allowed therapeutic approaches to be developed in which T cells mediating graft-versus-host disease after bone marrow transplantation can be selectively eliminated by the administration of ganciclovir. Although the strategy has been shown to be generally successful in early clinical trials, there are concerns about possible resistance to ganciclovir and the risk of myelosuppressive side-effects at the doses required to induce T cell suicide. We have incorporated the enhanced mutant HSV-TKSR39 into retroviral vectors tailored to exhibit high levels of expression in T cells and have used protocols optimized for the transduction and selection of primary lymphocytes. We demonstrate that leukemic and primary T cells can be efficiently transduced and highly enriched under conditions that should be readily adaptable for clinical use. T cells carrying HSV-TKSR39 were inhibited by exposure to ganciclovir at concentrations an order of magnitude below those required for wild-type HSV-TK. The less toxic agent aciclovir also eliminated T cells transduced with HSV-TKSR39 (but not HSV-TK), underlining the increased therapeutic potential of the mutant suicide gene system in the bone marrow transplantation setting.

Protein assays for diagnosis of Wiskott-Aldrich syndrome and X-linked thrombocytopenia.

Mutations in the gene encoding the Wiskott-Aldrich syndrome protein (WASp) give rise to Wiskott-Aldrich syndrome (WAS), a condition that exhibits a wide spectrum of clinical severity. Patients may develop mild thrombocytopenia or suffer from a wide range of associated disorders including eczema, immune dysfunction, autoimmune disease and malignancy. The clinical diagnosis of Wiskott-Aldrich syndrome (WAS) can be difficult and is usually supported by the detection of WASp gene mutations using genetic analysis. Recently, protein-based assays have been used to demonstrate the absence of WASp in patients known to have WASp gene mutations. We have now reversed this approach and report on the use of immunoblot assays to rapidly diagnose WAS in 13 patients. There was a complete absence of WASp in 10 out of 13 patients and an abnormal protein form was detected in the remaining three patients. In all cases, subsequent genetic analysis confirmed the presence of a WASp gene mutation. We believe that protein-based assays should be employed as the first line of investigation in the diagnosis of WAS spectrum disorders.

Anticardiolipin antibodies and thromboembolism after BMT.

We describe a 4-month-old child who developed unusual thrombotic complications following allogeneic BMT for Omenn syndrome, a form of SCID. Eight weeks after the procedure the child suffered a major cerebrovascular accident and developed acute pulmonary hypertension in association with persistently elevated anticardiolipin antibody titres. It is postulated that central line-derived microemboli caused these serious thrombotic complications in the context of an evolving hypercoaguable state.

Does staphylococcal enterotoxin B bind directly to murine T cells?

We have investigated the binding potential of staphylococcal enterotoxin B (SEB) to murine T cells using the induction of early activation events in Th1 and Th2 T-cell clones in the absence of antigen-presenting cells (APC) as indicators of direct interactions between SEB and the T-cell receptor (TcR). We consistently found that concanavalin A (Con A) induced rises in intracellular free calcium as well as inositol phosphate accumulation in APC-free T-cell clones. However, SEB uniformly failed to induce either calcium fluxes or inositol phosphate turnover in Th1 and Th2 T-cell clones in the absence of APC. In addition, we have used proliferation assays to show that (i) T-cell clones prepulsed with SEB did not respond when APC were added, (ii) APC-independent T-cell clones responded to soluble anti-TcR antibodies but not to SEB in the absence of APC, and (iii) SEB coupled to Sepharose beads did not stimulate T-cell clones in the absence of APC. Taken together our results argue against SEB binding to the TcR without the participation of MHC class II molecules.