Detection of adeno-associated virus type 2 in sorted human bone marrow progenitor cells.

Wild-type adeno-associated virus (wtAAV) is a helper-dependent human parvovirus which has the ability to integrate into the genome of a wide variety of human cells, including those of the hemopoietic lineages. Recombinant adeno-associated virus (rAAV) is becoming a good candidate for virally mediated gene therapy. rAAV is likely to be a safe vector in clinical gene transfer, as it has never been associated with any disease despite previous studies showing that up to 70% of adults are seropositive for wtAAV. Seroconversion appears to occur early in life. wtAAV is an upper respiratory tract virus that is gut secreted, but little is known about the integration of latent wtAAV in hemopoietic lineages. Unlike retroviruses, which have been the most common vehicles for gene transfer to date, wtAAV appears to have a preferred integration site in the target cell which has been termed AAVS1. Several studies have shown that wtAAV can only integrate into only one of the pair of chromosome 19 in a cell. This may have implications for the use of rAAV in gene transfer because patients with latent virus would be refractory to further infection with rAAV. We used a polymerase chain reaction (PCR) assay to detect the presence of wtAAV in the bone marrow samples from 106 patients who presented at our institution. We were able to detect the presence of integrated virus in 18 whole marrow samples. Subsequently CD34+ and CD3+ cell subsets were sorted from the cryopreserved marrow of three PCR-positive individuals to assess integration of virus in these cell lineages. In all three samples tested, we were unable to detect wtAAV virus in the CD34+ hematological precursor cells, but a detectable level of integrated viral DNA was demonstrated in the CD3+ cell fraction. Our findings therefore suggest that CD34+ cells might remain a good target for rAAV-mediated gene transfer despite previous wtAAV infection.

The effect of T cell depletion with Campath-1M on immune reconstitution after chemotherapy and allogeneic bone marrow transplant as treatment for leukaemia.

The prophylactic use of T cell depletion (TCD) strategies for the prevention of graft-versus-host disease (GVHD) following allogeneic stem cell transplantation remains widespread. Initial reports of high incidence of graft rejection after TCD BMT led to a move away from this approach but improved conditioning regimens have reduced this risk substantially. The use of TCD has also been associated with higher relapse risk post-BMT although the success of donor leukocyte infusion (DLI) as treatment for relapse has reduced this problem, especially in chronic myeloid leukaemia (CML). Currently the use of TCD BMT is increasing particularly due to the relative increase in BMT from non-related donors for whom TCD is the optimal GVHD prophylaxis. However, doubts remain over the long-term effect on the reconstituted immune system of recipients of TCD BMT, particularly in adult recipients. In this study we have undertaken a detailed sequential analysis in 23 patients who received allo-grafts from HLA-identical sibling donors after high-dose chemo/radiotherapy for acute or chronic leukaemia. Of these patients, 11 received non-manipulated grafts, five received 'partially TCD' (PTCD) and a further seven received 'fully TCD' (FTCD) bone marrow. T cell depletion was performed ex vivo by Campath-1M plus autologous serum as a source of complement. Partial TCD describes grafts with a T cell reduction of 1-2 log. Full TCD refers to grafts with a reduction of >2.5 log. The decision regarding the optimal degree of TCD was clinical and was based upon the perceived relative risk of relapse based upon the disease and remission status. All patients were monitored for up to 12 months post-BMT with regard to reconstitution of T and NK cell subsets. T cell depletion at either level was associated with a slower recovery of CD4 cells. This was most marked in the FTCD recipients and lasted throughout the period of study. CD8 cell recovery was also slower in the TCD recipients but this normalised throughout the 12 months post-BMT. The ratio of CD45RA+:CD45RO+ increased in all recipients after month 3. This suggests that a degree of extra-thymic T cell maturation can occur in recipients of allogeneic BMT. NK cell recovery was more rapid in the TCD recipients and these differences were maintained throughout the first year.

Intracellular T lymphocyte cytokine profiles in the aqueous humour of patients with uveitis and correlation with clinical phenotype.

This study aimed to investigate whether T cells in aqueous humour are different in different types of uveitis and correlate with clinical phenotype. Patients with clinically different types of uveitis, but all displaying active anterior uveitis, were phenotyped and samples of aqueous humour (AH) and peripheral blood (PB) collected. Cells from AH and PB were separated by centrifugation and by density gradient centrifugation (to obtain mononuclear cells PBMC), respectively. Cells were activated with PMA and ionomycin in the presence of Brefeldin A, stained for surface markers and intracellular cytokines, and analysed by flow cytometry. The cytokine profile was correlated with the clinical phenotype. Increased percentages of interleukin (IL)-10+-, but not interferon (IFN)-gamma+ T lymphocytes were found in AH compared with PB in patients with acute anterior uveitis (AAU), FHC or chronic panuveitis (PU). There was a trend towards elevated levels of IL-10+ T cells in AH from patients with FHC compared with AH from acute uveitis and panuveitis patients. Increased levels of IL-10+ T cells in AH compared with PB were also found in samples from patients with isolated uveitis, but not those with associated systemic disease. Levels of cytokine-positive T cells were not associated with the use of topical steroids or to the severity of the anterior uveitis. While type I cytokine-producing T lymphocytes are present in AH during AU, the presence of increased proportions of IL-10+ T lymphocytes in AH from patients with uveitis may be indicative of an anti-inflammatory mechanism that may influence the type and course of ocular inflammation in these patients.

Possible mechanism of selective killing of myeloid leukemic blast cells by lymphokine-activated killer cells.

Major histocompatibility complex-unrestricted lymphokine-activated killer (LAK) cells have been proposed as therapy for a variety of hematologic malignancies. Because these cells recognize and kill their targets independently of their antigen specific CD3 receptor, it is unclear how they might discriminate between normal and malignant cells. We now propose one such mechanism for the selective killing of myeloid leukemia blasts. While both CD2+ and CD2- activated killer cells may inhibit the clonogenic growth of myeloid leukemia cells, only the CD2+ subset effectively inhibits the growth of normal myeloid (granulocyte-macrophage and granulocyte) progenitors. This difference appears to reflect differential requirements for cell adhesion molecule recognition between normal and malignant progenitor cells. Inhibition of the growth of normal granulocyte-macrophage colonies by CD2+ LAK cells is blocked by antibodies to the CD2-lymphocyte function-associated antigen 3 (LFA-3) (CD58) cell adhesion system. In contrast, these antibodies have no effect on CD2+ LAK-mediated inhibition of malignant cell clonogenic growth. Instead, antibodies to the LFA-1 (CD11a/CD18)-intercellular adhesion molecule 1 (ICAM-1) (CD54) adhesion system reduce inhibition. These differences correspond to differential expression of the CD54 cell adhesion molecule by normal and malignant myeloid progenitor cells because less than 15% of normal CD34 positive cells are CD54+ while greater than 85% of CD34+ acute myeloid leukemia blasts express the CD54 antigen. LFA-3, the ligand for CD2, is strongly expressed by erythrocytes, and these cells competitively inhibit killing of normal but not malignant clonogenic cells in an analogous way to the effects of monoclonal antibody to the CD2-LFA-3 adhesion system. The operation of this effect in vivo may be a basis for selective cytotoxicity by CD2+ LAK against clonogenic myeloid blast cells, and could be exploited further with infusion of appropriate monoclonal antibodies.