Summary:immune mediated. This is evidenced by the observations of a reduced risk of leukaemia relapse after allogeneic BMT Anti-leukaemia activity after allogeneic bone marrow when compared with autologous or syngeneic BMT. Furtransplantation has been studied extensively but its antithermore, an increased incidence of leukaemic relapse has gen specificity and effector cell phenotype remain been reported after aggressive GVHD prophylaxis with unknown. Here we report a study in three recipients of cyclosporin A, 1 or lymphocyte depletion. The association autologous bone marrow transplantation done as part between GVHD and the graft-versus-leukaemia activity of of the treatment for acute leukaemia, in whom we were allogeneic BMT is strong and has led to a number of workable to detect innate specific anti-leukaemia activity ers concluding that GVL might be inseparable from GVHD. post-transplant. One patient maintained selective cellHowever, the lymphocyte subsets responsible for GVHD mediated cytolytic activity against her autologous leuand GVL remain to be elucidated in man despite intensive kaemic cells in the absence of cytolysis of her normal efforts. leukaemia (M5). A second patient was trans-Studies of GVL in the allogeneic setting are beset with planted for acute lymphoblastic leukaemia and had the problem of alloreactivity against major and minor histodetectable anti-leukaemia activity up to 20 weeks postcompatibility antigens. A number of studies have been per-ABMT. At this point anti-leukaemia activity could no formed in the autologous setting but none have reported longer be demonstrated and the patient suffered a evidence of innate cytotoxic activity which is specific for relapse 2 weeks later. A third patient was transplanted autologous leukaemic cells. Lotzova and colleagues 6 demfor AML (M4 Eo) and lacked detectable leukaemiaonstrated that patients prior to chemotherapy had low sponspecific immune reactivity at 1, 3 and 6 months posttaneous NK activity against the erythroblastoid leukaemic ABMT. She relapsed 6 months after her ABMT and cell line K562 and no detectable activity against autologous returned to complete remission after further chemoleukaemic cells. However, both were enhanced following therapy. She commenced treatment with alpha interin vitro culture with IL-2, although the leukaemia-speciferon and regained NK function. Furthermore, she ficity of the activity remains unknown since cytotoxicity of developed high level cytolytic activity against her autoautologous normal myeloid cells by the IL-2 stimulated logous leukaemic cells in the absence of activity against cells was not studied. A recent study of CLL patients after her remission bone marrow samples. She remains in fludarabine treatment demonstrated recovery of NK cell complete remission 17 months after her initial relapse.killing of K562 targets to normal levels and, in three of This is the first report of an apparent association five patients, a specific lytic action against their autologous between in vitro leukaemia-specific cyt...

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.

Low expression of CD45RB on CD45RO+ T lymphocytes defines a subset of highly differentiated T lymphocytes that accumulate in vivo within the affected joints of patients with rheumatoid arthritis (RA). Although it is known that CD45RO+ T lymphocytes migrate to sites of inflammation in vivo, it is not clear whether within this subset the CD45RBlo cells are selectively recruited or develop in situ within the joint. Using a transwell system we show that a small proportion of resting T lymphocytes migrated across unactivated human umbilical vein endothelial cells (HUVEC). These migrating cells were CD45RO+ and enriched for low CD45RB expression. In addition, both the CD45RO+CD45RBlo subset and migrating cells expressed increased levels of β1 and β2 integrins and CD44. The percentage of CD45RO+CD45RBlo T lymphocytes was increased in the circulation of patients with acute Epstein–Barr virus (EBV) infection. These in vivo activated cells also expressed increased levels β1 and β2 integrins and CD44, and showed an enhanced rate of transmigration compared with resting T lymphocytes. Transmigration of T lymphocytes was increased using the chemokines RANTES and lymphotactin and the cytokine interleukin‐15 (IL‐15). In addition, infection of the HUVEC with cytomegalovirus (CMV) led to an enhanced movement of T lymphocytes. In all of these cases the selective migration of the CD45RBlo subset was maintained. Thus although the rate of T‐lymphocyte transmigration could be influenced by a number factors, the CD45RO+CD45RBlo subset has a migratory advantage suggesting that more differentiated CD45RO+CD45RBlo T lymphocytes are selectively recruited to sites of inflammation.

We used an optical biosensor to determine the relative binding affinity of peptides to purified HLA class I molecules. In this assay we monitor beta2-microglobulin (beta2m) exchange within the HLA-A2 molecule, whereby native beta2m in the complex is replaced by beta2m immobilized at the surface of the biosensor. Quantitative kinetic measurements permit us to obtain association rate (kass), dissociation rate (kdiss) and affinity constants (KA) for the beta2m exchange reaction, alone, (control) and in the presence of exogenous peptide. We tested a panel of six peptides which had been designed and synthesized with an HLA-A2 binding motif, and had also been tested by the T2-cell binding assay, along with control peptides. The biosensor results demonstrate that exogenous peptide influences the dynamics of beta2m exchange in a sequence-specific manner. Five of six peptides increased the association rate, decreased the dissociation rate, and significantly increased the affinity (KA=1. 55-1.88x10(9) M-1) of HLA-A2 for immobilized beta2m compared with the control (KA =1.14+/-0.04x10(9)M-1), demonstrating stabilization of the complex. One peptide was unable to stabilize the complex, as also shown in the T2 binding assay. However, analysis of peptide sequences demonstrated that the HLA-A2 secondary motif as well as primary motif residues are required for HLA-A2 stabilization. Further experiments demonstrated that beta2m exchange alone cannot stabilize the HLA class I complex at the cell surface until a peptide of sufficient binding affinity is bound. Hence kinetics equal to or below the control values in our biosensor assay probably represent an unstable complex in vivo. Unlike other methods described for the analysis of peptide stabilization, this approach is significantly faster, provides full kinetic analysis, and is simpler, since it requires no labeling of peptides. Furthermore, this may have important implications in the assessment of peptide vaccines.

The evaluation of contaminating breast cancer cells in hematopoietic grafts is of considerable importance for monitoring the efficiency of purging procedures. We report a comparison of three systems for the in vitro detection and enumeration of metastatic breast cancer cells. Breast cancer cells from established cell lines were mixed with Daudi cells at dilutions ranging from 1:10 to 1:1,000,000, and a predetermined number were fixed in defined areas on microscope slides coated with one of the following attachment factors: (i) Cell-Tak Cell and Tissue Adhesive, (ii) 0.1% solution of Poly-L-Lysine, or (iii) Cel-Line HTC Super Cured slides. We employed a specificity-proven pancytokeratin antibody (A45-B/B3) and the alkaline phosphatase-antialkaline phosphatase (APAAP) staining technique. In multiple experiments, one breast cancer cell in 1,000,000 Daudi cells could reliably be detected in the Cell-Tak and Cel-Line systems and 1 in 100,000, with the Poly-L-Lysine system. The observed number of seeded cells showed a highly significant correlation with the number of cells seeded (p < 0.0001 in all cases). Finally, we used the Cell-Tak method to evaluate clinical material from various sources: from patients with primary carcinomas of the breast, prechemotherapy, and during various chemotherapeutic regimens, as well as from patients with metastatic disease. The system consistently detected tumor cells in bone marrow samples from these patients. All peripheral blood samples from patients with metastatic disease tested positive at incidences ranging from 5 to 19/10(6) peripheral blood mononuclear cells. This is a simple and reliable technique that allows rapid screening of large cell numbers with high resolution of positive cells.