Immunodeficiency after Allogeneic Bone Marrow Transplantation in Man

This study was undertaken to clarify the mechanism behind the severely decreased lymphocyte proliferative response upon stimulation with mitogens and antigens seen after allogeneic bone marrow transplantation (BMT) in man. We investigated eight BMT patients and eight controls and found that the proliferative response of patient cells was reduced both when the cells were stimulated with phytohaemagglutinin (PHA) and when they were stimulated with a combination of phorbol myristate acetate (PMA), which is an activator of protein kinase C (PKC), and the calcium ionophore A23187, which irreversibly opens for calcium transport into the cell (median relative responses were 41 and 37%, respectively). However, the PHA-induced increase in the concentration of intracellular free calcium in post-BMT cells was not significantly different from the values found in control cells and the expression of interleukin 2 (IL-2) receptors (CD25) was only slightly decreased. However, the production of IL-2 was severely decreased in patient cells after stimulation with A23187/PMA (median 3541 units), although it was higher than in PHA-stimulated control cells (median 354 units). These results show that a direct activation of PKC by PMA combined with an increase in intracellular free calcium by A23187 cannot overcome the lymphocyte proliferation deficiency in cells from patients after allogeneic BMT. The data suggests that the defect is affecting the diacylglycerol pathway considerably more than the inositol triphosphate pathway.     

Lymphocytes Bearing the γ/δ T-Cell Receptors in Down''s Syndrome

Subjects with Down's syndrome (DS), or trisomy 21, have an increased susceptibility to infections, malignant diseases and autoimmune phenomena. Various arms of the immune system are severely impaired in trisomic patients. We found that the proportion of blood lymphocytes bearing the gamma/delta T-cell receptor (TCR) was significantly higher in adults with trisomy 21 than in age- and sex-matched healthy controls. Interestingly, the increase was mainly due to an over-expansion of cells which bear non-covalently bound gamma/delta chains on their surface. This contrasts with the normal blood picture, where the great majority of gamma/delta T cells express the disulphide-linked form of the TCR. The fact that trisomic gamma/delta T cells are both numerically and phenotypically unbalanced provides further evidence that immunological abnormalities are integral features of DS.     

Structure of the T-Cell Receptor in a Tiαβ, Tiγδ Double Positive T-Cell Line

The multichain T-cell receptor is composed of at least six different polypeptide chains. The clonotypic Ti heterodimer (Tiαβ or Tiγδ) is non-covalently associated with the CD3 chains (CD3γδɛζ). The exact number of subunits constituting the T-cell receptor is still not known. It has been suggested that each T-cell receptor contains two Ti dimers. To gain insight into the structure of the T-cell receptor we constructed a Tiαβ, Tiγδ double positive T-cell line which contained four functional Ti chains (Tiαβ, β, γ, and δ). The data demonstrated an absence of Ti dimers containing mixtures of chains other than the typical Tiαβ and Tiγδ combinations. Furthermore, by co-modulation experiments we demonstrated that the Tiαβ and the Tiγδ dimers were not expressed in the same T-cell receptor. Our data indicate that the T-cell receptor does not contain two Ti dimers.     

Toll-Like Receptor Expression and Function in Subsets of Human γδ T Lymphocytes

Two subsets of human γδ T cells can be identified by T cell receptor (TCR) V gene usage. Vδ2Vγ9 T cells dominate in peripheral blood and recognize microbe- or tumour-derived phosphoantigens. Vδ1 T cells are abundant in mucosal tissue and recognize stress-induced MHC-related molecules. Toll-like receptors (TLRs) are known to co-stimulate interferon-γ (IFN-γ) production in peripheral blood γδ T cells and in Vδ2Vγ9 T cell lines. By microarray analysis, we have identified a range of genes differentially regulated in freshly isolated γδ T cells by TCR versus TCR plus TLR3 stimulation. Furthermore, we have investigated TLR expression in freshly isolated Vδ1 and Vδ2 subsets and cytokine/chemokine production in response to TLR1/2/6, 3 and 5 ligands. TLR1,2,6,7 RNA was abundantly expressed in both subsets, whereas TLR3 RNA was present at low levels, and TLR5 and 8 RNA only marginally in both subsets. Despite abundant RNA expression, TLR1 was rarely detectable by flow cytometry. In contrast, TLR2 and TLR6 proteins were detected in purified Vδ1 and Vδ2 T cells, and TLR3 protein was detected intracellularly in both subsets. TLR1/2/6, 3 and 5 ligands co-stimulated the IFN-γ and chemokine secretion in TCR-activated Vδ1 and Vδ2 subsets, although the levels of IFN-γ secreted by Vδ1 T cells were much lower than those produced by Vδ2 T cells. Our results reveal comparable expression of TLRs and functional responses to TLR ligands in freshly isolated Vδ1 and Vδ2 T cells and underscore the intrinsically different capacity for IFN-γ secretion of Vδ1 versus Vδ2 T cells.     

Different Activation States of Human Lymphocytes after Antibody-Mediated Stimulation via CD3 and the α/β T-Cell Receptor

BMA031 is an IgG2b antibody directed towards the human alpha/beta T-cell receptor that is able to induce proliferation of peripheral blood mononuclear cells independent of antibody crosslinking. The proliferative response to BMA031 during the first 3 days of culture is usually of similar magnitude to that induced by the IgG2a CD3 antibody OKT3 but decreases quickly afterwards. Stimulation by BMA031 induces no measurable IL-2 release, very low expression of the IL-2 receptor, and does not trigger cytotoxic effector function. However, cross-linking of the antibody or addition of IL-2 leads to enhanced and prolonged proliferation, strong IL-2 receptor expression, and cytotoxic activity, features that are usually found after stimulation by the IgG2a CD3 antibody OKT3 in soluble form. The stimulatory effect of BMA031 cannot be diminished by IL-2 receptor blocking, whereas stimulation by OKT3 is strongly reduced. Moreover, proliferation induced by BMA031 has lower sensitivity to inhibition by ciclosporin than OKT3. From these results two major conclusions can be drawn: (1) an IL-2-independent way of activation may be important for the short-term proliferation of the T cells stimulated by BMA031 and (2) after stimulation by BMA031, cells reach a state of activation that is different from that induced by OKT3. These differences are most likely related to the different specificities of the antibodies, alpha/beta TcR versus CD3, suggesting that different activation signals are triggered via CD3 and via the alpha/beta TcR.     

T Lymphocytes Bearing the γδ T Cell Receptor are Susceptible to Steroid-Induced Programmed Cell Death

The mechanisms by which glucocorticoids suppress immune responses have not yet been clearly defined. In steroid-sensitive pathological conditions, an increase in γδ T cells can occur in certain untreated systemic autoimmune disorders and seems to be a persistent feature in most cases of systemic lupus erythematosus (SLE). Our previously published data demonstrated that immunosuppressive therapy normalized this expanded SLE T cell subset in parallel with clinical remission of the symptoms. To establish how corticosteroid treatment determines the disappearance of peripheral blood γδ T lymphocytes, circulating αβ and γδ T lymphocytes from seven SLE subjects with active disease and seven healthy individuals were cultured in the presence or absence of 10_-7M Dexamethasone (DEX). Cell suspensions were then analysed for DNA fragmentation, characteristic of apoptotic cell death, by a new cytofluorimetric method. Conventional agarose-gel electrophoresis on the same T cell populations was carried out for comparison. Regular follow-ups for 6 months revealed in vivo steroid treatment determined a dramatic fall in SLE blood γδ T cells, and in vitro experiments seem to indicate that DEX-triggered apoptotic signals are confined to the double negative (CD4_- CD8_-) γδ T cell subpopulation which disappears after in vivo immunosuppressive therapy. Clinical and pathological remission of some autoimmune diseases is often obtained by corticosteroids. Our results offer new insights on the mechanisms through these hormones exert their potent inhibitory activities on immune system cells postulated to play a role in the generation of autoimmune responses.     

T-Cell Receptor Gene Expression by Human γδ T-Cell Clones from Peripheral Blood and Reproductive Tissues in Relation to Non-MHC-Restricted Cytotoxic Function

T-cell receptor gamma and delta gene expression was determined using V-region-specific monoclonal antibodies in conjunction with Southern blot analysis in panels of gamma delta T-cell clones from human peripheral blood (n = 77) and reproductive tissue (n = 9). Whereas 53 out of 77 (69%) clones from peripheral blood expressed V gamma 9 and V delta 2J1, only 2 out of 9 (22%) from reproductive tissues expressed V delta 2J1. Two out of eight decidual clones expressed both V gamma 9 and V delta 1J1, while this configuration was rare in clones from peripheral blood. The majority of clones from the peripheral blood of one donor expressed V gamma 8 and V delta 3J1. Clones were identified which expressed V delta 1J1 in the disulphide-linked C gamma 1 form of the receptor and which expressed a gene other than V delta 1 in the non-disulphide-linked C gamma 2 form, indicating incomplete concordance between expression of V delta 1 and C gamma 2. V delta 3 could be expressed in the disulphide-linked or non-disulphide-linked form of the receptor. At least 5 out of 77 peripheral clones were expressing V delta genes other than V delta 1, V delta 2, or V delta 3 in conjunction with C gamma 1 or C gamma 2. There was a strong but incomplete correlation between high non-NHC-restricted cytotoxic function and C gamma 1 expression. Clones from the same donor expressing both V gamma 9JPC gamma 1 and V delta 2J1 showed either high or negligible cytotoxicity, and cytotoxic clones expressing C gamma 2 were found. Thus no complete correlation between cytotoxic function and expression of a particular form of the gamma delta heterodimer was identified. The results also suggest that gamma delta T cells from reproductive tissues are less likely to express V delta 2J1 than those from peripheral blood.     

Rye γ-70 and γ-35 secalins and barley γ-3 hordein cross-react with ω-5 gliadin, a major allergen in wheat-dependent, exercise-induced anaphylaxis

Patients with wheat-dependent, exercise-induced anaphylaxis experience severe allergic reactions when exercising after ingestion of wheat. The major wheat allergen associated with these reactions is a omega-5 gliadin, and patients following a gluten-free diet have remained free of symptoms. The aim of this study was to examine whether allergens cross-reacting with wheat omega-5 gliadin are present in rye, barley and oats. Sera from 23 adult patients with wheat-dependent, exercise-induced anaphylaxis were examined. Cereal allergens cross-reacting with wheat omega-5 gliadin were identified by immunoblot inhibition. The cross-reactive allergens were purified by gel filtration and reversed-phase chromatography and submitted to amino acid sequencing. Cross-reactivity was further studied by IgE ELISA and ELISA inhibition, and in vivo reactivity by skin prick testing. In immunoblotting rabbit anti-omega-5 gliadin antibodies bound to 70 kDa and 32 kDa proteins in rye and a 34-kDa protein in barley, but not to proteins in oats. N-terminal sequencing identified these proteins as rye gamma-70 secalin, rye gamma- 35 secalin and barley gamma-3 hordein, correspondingly. In ELISA 21/23 (91%) patients with wheat-dependent, exercise-induced anaphylaxis showed IgE antibodies to purified gamma-70 secalin, 19/23 (83%) to gamma-35 secalin and 21/23 (91%) to gamma-3 hordein. In ELISA inhibition omega-5 gliadin inhibited over 90% of the IgE binding of pooled patient sera to solid-phase gamma-secalins and gamma-3 hordein. Skin prick testing gave positive reactions to gamma-70 secalin in 10/15 (67%) patients, to gamma-35 secalin in 3/15 (20%) patients and to gamma-3 hordein in 7/15 (47%) patients. The results of this study show that gamma-70 and gamma-35 secalins in rye and gamma-3 hordein in barley cross-react with omega-5 gliadin, a major allergen in wheat-dependent, exercise-induced anaphylaxis. These findings suggest that also rye and barley may elicit symptoms in patients with wheat-dependent, exercise-induced anaphylaxis.     

A Minority of Patients with Rheumatoid Arthritis Show a Dominant Rearrangement of T-Cell Receptor β Chain Genes in Synovial Lymphocytes

The clonality of T lymphocytes isolated from the synovial fluid and peripheral blood of patients with rheumatoid arthritis was investigated by restriction enzyme fragment mapping of the rearrangements of the beta chain gene of the T-cell antigen receptor. Three patients showed a dominant rearrangement amongst their synovial fluid T cells which was not seen in their peripheral blood T-cell population, suggesting the presence of a predominating T-cell clone. However, most of the patients examined (8 out of 11) demonstrated polyclonal T-cell populations in both their synovial fluid and peripheral blood. Of four synovial fluid T-cell lines investigated, one showed evidence of a dominant T-cell clone.     

Acceleration of Immune Reconstitution after Bone Marrow Transplantation in Mice by Bone Marrow Stromal Cell Line Transfected with IL-6 Gene

After bone marrow transplantation, dysfunction of immunesystem is a critical problem to be resolved in clinical treat ment. Efficient bone marrow transplantation needs com pleted immune reconstitution, which is associated to manyfactors. Among them, Chemo/radiotherapy after and/orbefore BMT can cause great damage to hematopoietic mi croenvironment, which decrease the ability of proliferationand differentiation of stem cell[1]. To observe the effectof bone marrow stromal cell on the imm…     

The Interactions of γδ T Cells with Extracellular Matrix: Receptor Expression and Utilization Patterns

Purified populations and clones of human γδ T cells were examined for their ability to interact with extracellular matrix (ECM) components. The stimulation of these cells with phorbol ester induced cellular adhesion for ECM, The adhesion structures for fibronectin and collagen were shown to be members of the CD29 integrin family. The expression patterns of β1, β2 and β3, integrin-s by these cells were examined. The receptor expression and utilization patterns suggest that αβ, γδ T cells and B cells have similar repertoires of adhesion structure.     

The Effect of Natural Killer Cell Killer Ig-Like Receptor Alloreactivity on the Outcome of Bone Marrow Stem Cell Transplantation for Severe Combined Immunodeficiency (SCID)

Natural killer (NK) cell alloreactions against recipient cells in the setting of bone marrow transplantation have been associated with decreased rates of graft-versus-host disease (GVHD) and improved survival in transplant recipients with myeloid leukemia. These alloreactions are predicted by the absence of recipient HLA class I ligands for donor inhibitory killer Ig-like receptors (KIR). We hypothesized that donor NK cell alloreactions against recipient cells may affect the development of T and B-cell functions and incidence of GVHD in infants with severe combined immunodeficiency (SCID). Of the 156 patients with SCID who had received related bone marrow transplants without pretransplant chemotherapy or posttransplant GVHD prophylaxis, 137 patient-donor pairs were evaluated for the absence of recipient HLA class I ligands for donor inhibitory KIR. Analysis showed that the absence of a KIR ligand had no effect on the incidence or severity of GVHD (RR [corrected] = 0.95, p = 0.84), development of T-cell function (RR [corrected] = 1.05, p = 0.69), production of IgA (p = 0.46) or IgM (p = 0.33), or on 5-year survival (RR [corrected] = 1.21, p = 0.10). Further, in patients possessing native NK cells, the absence of KIR ligands in donors for recipient-inhibitory KIR did not alter transplantation outcomes. This study suggests that inhibitory KIR/HLA interactions do not play a significant role in bone marrow transplantation for SCID.     

HLA-Haploidentical Donor Lymphocyte Infusions for Patients with Relapsed Hematologic Malignancies after Related HLA-Haploidentical Bone Marrow Transplantation

Treatment of relapse after related HLA-haploidentical T cell–replete bone marrow transplantation (haploBMT) with post-transplantation cyclophosphamide (PTCy) using haploidentical donor lymphocyte infusion (haploDLI) is not documented. All patients who received haploDLI after haploBMT with PTCy between June 2003 and October 2012 were identified and assessed for graft-versus-host disease (GVHD) and outcomes. Forty patients received 52 haploDLI doses. Sixteen patients had acute myeloid leukemia, 11 had lymphomas, and 34 had nonmyeloablative conditioning before haploBMT. The median time from haploBMT to relapse was 183 (range, 0 to 1399) days. The median age at haploDLI was 48 (range, 3 to 70) years. The first haploDLI doses were 1 × 10⁵ CD3⁺ cells/kg with subsequent escalation. The most commonly used first haploDLI dose was 1 × 10⁶ CD3⁺ cells/kg. The median follow-up after haploDLI was 7 (mean, 15.4; range, .5 to 96) months for the entire cohort, and 17.5 (mean, 28; range, 2.4 to 96) months for the responders. Acute GVHD developed in 10 patients (25%), 6 patients had grade 3 to 4, and 3 developed chronic GVHD. Twelve (30%) patients achieved a complete response (CR) with a median duration of 11.8 (mean, 22.5; range, .4 to 94) months. At last follow-up, 8 responders were alive in CR; 6 for over a year. HaploDLI for relapse after haploBMT is associated with acceptable toxicities and can result in durable responses.