A human monoclonal antibody reacting against HLA-DQ1-, DQ4-, and a subset of DQ7-bearing cells.

Human mAb 2A2 recognizes an epitope present in the HLA-DQ1 + 4 specifications and also on several DQ7-positive cells. We have investigated the extra reactions of this monoclonal reagent on a wider panel of DQ1-, DQ4-negative/DQ7-positive B-cell lines. The results obtained support the existence of two subtypes of the HLA-DQ7 specificity on the basis of their reactivity with human mAb 2A2; the DQ7/2A2-positive variant has been found in 12 of 29 BCLs positive for the DR11 antigen, and in four of eight BCLs bearing DR4-DQ7 haplotypes. It has also been detected in the DR12-positive cells assayed and in several unusual DR/DQ7 combinations not commonly found in Caucasoid populations, including the DR13-DwHAG and DR14-Dw16 haplotypes. Results from competition binding assays between 2A2 and well-characterized murine anti-DQ polymorphic mAbs suggest that the epitope recognized by human mAb 2A2 on DQ1- or DQ4-bearing haplotypes is located on the DQ beta chains of such specificities, being amino acid residues 54-55, the potential binding site of antibody 2A2, whereas the binding site on DQ7 antigens cannot be explained on the basis of known amino acid sequences.

The placental blood program of the Barcelona Cord Blood Bank.

Cord blood has recently become an alternative to bone marrow transplantation, generating the need for cord blood banks where large numbers of frozen cord blood units can be stored. The Barcelona Cord Blood Bank (bcB) was created in October 1995. Initially, several methods for volume reduction were tested, including Ficoll, Percoll, Gelatin and HES sedimentation. Of these, HES sedimentation (88% +/- 11 CD34+ cells recovery) was the one chosen for routine banking. Up to November 1997, the bank has processed 754 units with a median of 1.05 x 10(9) nucleated cells and 2.5 x 10(6) CD34+ cells stored per unit. Nine of these units have been shipped for transplantation.

Differential responsiveness of human B lymphocytes to phorbol ester and calcium ionophore based on their state of activation.

For tonsil B cells of a particular high density (below 65% Percoll), both phorbol myristate acetate (PMA) (5 ng/ml) and calcium ionophore A23187 (500 nM) were required to induce RNA synthesis, significant DNA synthesis also occurring in the presence of 12,000 MW B-cell growth factor (BCGF). In contrast, PMA alone, even at 1 ng/ml, was a sufficient stimulus to induce strong DNA synthesis in low-density B cells (45-50% Percoll) and strong proliferative responsiveness to BCGF in intermediate-density B cells (55-65% Percoll). In these latter B-cell populations, A23187 (500 nM), acted synergistically with non-mitogenic PMA doses to induce strong DNA synthesis, the PMA dose required being 5-50 times lower in low-density B cells (0.1-1 ng/ml) than in intermediate-density B cells (5 ng/ml). Preactivation for 30 hr with anti-Ig antibodies plus BCGF, known to drive B cells into late G1, rendered high-density B cells responsive to PMA (1-10 ng/ml) with high, dose-related DNA synthesis. These data indicate that the B-cell mitogenicity of a given nanomolar dose of PMA depends on the more advanced state of activation of B cells. It was also found that the above optimal dose of A23187 (500 nM) paradoxically inhibited the PMA-induced DNA synthesis of low-density B cells and in vitro preactivated high-density B cells. Data obtained with low-density B cells suggest that a calcium influx during the PMA-induced proliferative phase of B cells may provide a negative signal for the DNA synthesis.

Cellular activation without proliferation to B cell growth factor and interleukin 2 in chronic lymphocytic leukaemia B cells stimulated with phorbol ester plus calcium ionophore.

Individual leukaemic B cells of chronic lymphocytic leukaemia (CLL) do not proliferate to B cell growth factor (BCGF) or interleukin 2 (IL-2) when co-stimulated with immunoglobulin (Ig) ligands. To exclude possible defective signalling via surface Ig (sIg), phorbol myristate acetate (PMA) plus calcium ionophore (A23187) were used to activate purified CLL B cells and compared with staphylococcal protein A coupled to sepharose beads (Seph-PA). RNA synthesis and phenotypic changes after PMA plus A23187 stimulation indicate that CLL B cells from (10) different individuals are similarly able to undergo the G0 to the G1 phase transition and express surface activation antigens. In contrast, they are variable in the capacity to show DNA synthesis, which occurred in only six out of 10 cases. Even in the presence of BCGF (10%, v/v) or IL-2 (50 U/ml) four out of nine CLL B cells activated with PMA plus A23187 or PMA alone were still unable to proliferate although they were induced to express CD23, 4F2, CD25 and OKT9 antigens by PMA plus A23187. However, PMA plus A23187 induced IgM secretion which increased further in response to IL-2 even in the absence of DNA synthesis. Moreover, in other CLL B cell populations, the unresponsiveness to growth factors upon co-stimulation with Ig ligands (Seph-PA) may simply reflect a defective signalling via sIg cross-linking which can be circumvented by PMA plus A23187 stimulation. Recombinant Interferon-gamma (50 U/ml) failed to affect DNA synthesis and IgM secretion.

Inhibitory effect of IL-4 on the sepharose-CD3-induced proliferation of the CD4CD45RO human T cell subset.

CD45R monoclonal antibodies are able to distinguish two different subsets of the CD4 human T cells. This phenotypic split is accompanied by functional diversity. In this report we have analyzed the capabilities of CD45R subsets of CD4 human T cells to use interleukin 2 (IL-2) and IL-4 as growth factors. We have found that both cell subsets are able to proliferate after stimulation with Sepharose-CD3 in the presence of externally added IL-2 or IL-4. However, the response to IL-4 of CD4CD45RO cells was comparatively lower than the response of CD4CD45RA cells. Both cell subsets showed a good response to Sepharose-CD3 plus adherent cells (AC), but when IL-4 was present in the culture only the CD4CD45RA cells showed an enhancement in the Sepharose-CD3-induced proliferation, while proliferation of the CD4CD45RO T cell subset was inhibited. Similar effects were seen, however, in the response to CD4CD45RA or CD4CD45RO cells to Sepharose-CD3 plus IL-2. Although the precise mechanism of the inhibitory effect of IL-4 is not known, the results obtained suggest that IL-4 could interfere in some way with the signalling of IL-2 to the proliferation of the CD4CD45RO T cell subset.

[Post-transfusion purpura. Description of 2 cases diagnosed at the same hospital in a 6-month period]. / Púrpura postransfusional. Descripción de dos casos diagnosticados en un mismo hospital en un periodo de seis meses.

Two new cases of postransfusional purpura diagnosed at the same hospital within the space of six months are described. This finding supports the idea that despite being an infrequent disorder, a substantial rise in PTP cases inside and outside our country, has been recorded. This increase has coincided with a greater interest in platelet immunology and, particularly, in complications associated with blood transfusion. Both cases constitute an example of the clinical epidemiological profile which characterizes the patients suffering from this disorder. The patients are two women aged 74 and 60 years who after 8 and 9 days, respectively, of being transfused with red cells developed a severe thrombocytopenia accompanied by generalized haemorrhagic diathesis. The serological studies performed revealed the presence, in both patients, of an HPA-1a platelet specific antibody. The platelet genotyping enabled us to confirm this specificity after detecting an HPA-1 (a-b+) platelet genotype. The treatment with immunoglobulins at high doses proved to be effective in both cases. The adsorption-elution experiments of the antibody versus HPA-1 (a + b) platelets were positive in the patient with the highest antibody titre (1024). This finding support the most recent hypothesis concerning the pathogenic mechanism of PTP. According to this theory, the antibody, which is detected in the acute phase of the PTP, would not yet have acquired the restricted specificity corresponding to it. This could enable it to react with a structure shared by the HPA-1a positive and HPA-1a negative platelets.

IL-4 inhibits IL-2 synthesis and IL-2-induced up-regulation of IL-2R alpha but not IL-2R beta chain in CD4+ human T cells.

There is growing evidence to suggest a regulatory role of IL-4 in the immune system affecting both proliferation and lymphokine production. In the present work we have analyzed the effect of IL-4 on IL-2 and IFN-gamma synthesis by stimulating CD4+ human T cells (+10% accessory cells) with Con A in the presence of several doses (1 to 100 U/ml) of human rIL-4. The results showed an impaired IL-2 and IFN-gamma synthesis in the presence of IL-4. This inhibition was dose dependent and was evident only when IL-4 was added in the first 2 h of culture. Moreover, the external addition of IL-2 did not revert the inhibitory effect of IL-4 on IL-2 and IFN-gamma synthesis induced by Con A. We have also analyzed the effect of IL-4 on the expression of both alpha- and beta-chains of the IL-2R. Although the expression of IL-2R alpha mRNA was not modified after 6 h in culture in the presence of IL-4, a decrease was observed at 24 and 48 h. The addition of rIL-2 showed that the inhibition in IL-2R alpha expression could be explained by an impairment in the up-regulatory signal transmitted through the IL-2R. In addition to this, IL-4 did not modify the IL-2R beta mRNA expression at 6 and 24 h although a decreased expression was observed at 48 h which could be explained by the defective IL-2 production. The differential effect of IL-4 on the up-regulatory effect of IL-2 in the expression of IL-2R alpha and IL-2R beta suggest the existence of different regulatory mechanisms acting on the expression of both chains.

Stimulation through the TCR/CD3 complex up-regulates the CD2 surface expression on human T lymphocytes.

Despite the well known interrelationship between the CD2- and CD3-mediated signal transduction pathways, it is not well established whether the CD2 surface expression can be regulated by triggering of TCR/CD3 complex. In this study we show that the stimulation of human PBMC with the Cris-7 (CD3) mAb, both in soluble and particulate form, results in hyperexpression of the CD2 surface Ag, as assessed by immunofluorescence and semi-quantitative immunoprecipitation assays. Similar effects on CD2 surface expression were obtained when different CD3 mAb (OKT3, RW2-8C8 and Leu-4) were tested. The CD3-mediated CD2 up-regulation was suppressed by cycloheximide and actinomycin D, indicating that it requires de novo protein and RNA synthesis. In agreement with this, increased CD2 RNA levels were observed after 3 h of stimulation, reaching a plateau at 24 h that was maintained for 72 h. The CD2 up-regulation was concomitant to other CD3-induced activation-related events such as induction of surface CD25 and CD71 and high RNA levels for c-myc, IL-2R alpha- and beta-chains, CD71, and IFN-gamma. CD2 up-regulation appeared to be elicited by a protein kinase C-dependent mechanism because it was abrogated by staurosporine, a potent protein kinase C inhibitor. Moreover, IL-2-dependent events may also help in enhancing CD2 hyper-expression because it was only partially inhibitable by cyclosporine, dexamethasone, or Mar-108 (CD25) mAb. In conclusion, our data suggest that CD2 up-regulation can be a relevant event in T cell activation triggered by the physiologic engagement of the TCR/CD3 complex.