Locomotor properties of human germinal centre B cells: activation by anti-CD40 and IL-4 allows chemoattraction by anti-immunoglobulin.

The locomotor properties of B cells isolated from the germinal centres (GC) of human tonsils were studied using polarization, collagen gel invasion and micropore filter assays. The proportion of motile GC cells in the freshly isolated population was small. During culture in interleukin-4 (IL-4)+anti-CD40, but not in control medium, the proportion of polarized cells increased and these cells migrated actively into collagen gels. After 24 hr culture, most of the surviving population was in locomotor morphology. The locomotor population consisted mainly of centrocytes in the G1 phase of growth. More locomotor cells than spherical cells took up [3H]uridine, but locomotor cells did not take up [3H]thymidine. After culture for 6 hr in IL-4+anti-CD40, GC B cells were tested in short-term polarization assays and filter assays for their response to chemoattractants. In both assays, a proportion of the cells responded to anti-IgA and to anti-IgA F(ab')2 fragments at 1 ng/ml., or to anti-IgG, anti-IgM and F(ab')2 fragments of these antibodies at 100 ng-1 microgram/ml. A checkerboard filter assay showed a good chemokinetic response and a weaker chemotactic response of GC cells to anti-IgA. Expression of Fc gamma RII (CD32) was increased after culture in IL-4+anti-CD40, and these cultured cells responded in filter and polarization assays to anti-CD32. Thus culture in IL-4 and anti-CD40 not only rescues GC B cells, but also increases their locomotor capacity and allows them to respond in chemotaxis assays to anti-immunoglobulin.     

Suppressor T cell induction by soluble OKT3 antibody does not require cross-linking of the T3 molecule

The monoclonal antibody OKT3 can induce T suppressor cells of the in vitro antibody response when used at concentrations as low as 10 ng/ml and in cultures containing as few as 4% OKT3-pretreated T cells. These suppressor cells are radioresistant, cyclosporin sensitive and belong to the T4 as well as the T8 subpopulations. In contrast with the mitogenic effect of OKT3 the suppressor activity can be induced and expressed in highly monocyte-depleted cultures. Moreover F(ab')2 fragments of OKT3, although not mitogenic, are able to induce suppressor cells. Thus cross-linking of the T3 molecule (such as that realized when intact OKT3 IgG and monocytes are present) is not required to induce suppressor cells among resting normal T cells.     

A noncognate interaction with anti-receptor antibody-activated helper T cells induces small resting murine B cells to proliferate and to secrete antibody

Culture of small resting allogeneic B cells (of an irrelevant haplotype) with two clones of T helper (Th) cells that were activated by the F23.1 anti-T cell receptor antibody led to the activation of B cells to proliferate and to secrete antibody. Th cell supernatants by themselves had no effect on resting B cells (even in the presence of intact F23.1 antibody), but could induce antibody secretion by anti-Ig-preactivated B cells. Both F23.1+ clones (E9.D4 and 4.35F2) and one F23.1- clone (D2.2) could synergize with supernatants from activated E9.D4 T cells to induce B cell activation. F(ab')2 fragments of F23.1 induced E9.D4 to activate B cells as efficiently as intact F23.1 and B cell populations that had been incubated with F23.1 were not activated when cultured with E9.D4, although T cells recognized cell-presented F23.1 and were weakly activated. Reduction of the density of F23.1 adsorbed to plastic resulted in weak T cell activation, and these T cells did not induce B cell responses. Haptenated B cell populations, although recognized by E9.D4, were not activated. Separation of T and B cells by a 0.4-micron membrane prevented T-dependent B cell activation, although Th cell-derived B cell-activating lymphokines would be assayed across these membranes. These results suggest a polyclonal noncognate B cell activation that depends on physical contact between B cells and activated T cells. The requirement for a cognate interaction of Th with B cells for the production and delivery of B help can therefore be overcome by activating Th cells with high densities of T cell receptor ligands.     

Modulation of type II Fc gamma receptor expression on activated human B lymphocytes

We have monitored Fc gamma RII expression during the activation of human B lymphocytes by simultaneous analysis of monoclonal antibody (mAb) binding and EA rosetting. The expression of Fc gamma RII showed a biphasic time course. Initially, a transient increase of Fc gamma RII with no ligand-binding capacity was observed with mAb staining as early as 10 min after stimulation by the F(ab')2 fragment of anti-human IgM or phorbol 12-myristate 13-acetate and then after 3 to 24 h a decrease in the number of Fc gamma RII+ cells was seen. Trypsin-like serine protease activity also appeared in the lysate of activated B cells at this time. On the 2nd day of activation a significant enhancement of Fc gamma RII expression was observed, mainly on enlarged blast cells as monitored by both mAb and by ligand binding (EA rosette). At the same time, soluble fragments of Fc gamma RII with the ability to bind human Fc were detected in the supernatant of activated B cells, probably as a result of proteolytic cleavage. These findings suggest that activated B cells are identical with the population of mononuclear cells which shed Fc gamma R when incubated at 37 degrees C. The ability of activated but not resting B cells to release Fc gamma RII correlates with the expression of early activation markers and with the appearance of a trypsin-like serine protease activity of the same cells; thus, the release of Fc gamma RII occurs in the early G1 phase of cell cycle as a result of proteolysis. Later the release of Fc gamma RII is accompanied by the enhancement of Fc gamma RII expression before the cells reach the S phase. The fragments of cleaved Fc gamma RII had an apparent molecular mass of 33 and 14-18 kDa under nonreducing conditions, and upon reduction fragments of smaller size were observed.     

F(ab')2 anti-CD4 and intact anti-CD4 monoclonal antibodies inhibit the accumulation of CD4+ T cells, CD8+ T cells, and B cells in the kidneys of lupus-prone NZB/NZW mice

Murine lupus in NZB/NZW (B/W) mice is characterized by immune-complex glomerulonephritis and lymphocytic infiltration of several organs, including the kidney. We recently showed that treatment of B/W mice with F(ab')2 anti-CD4 monoclonal antibody retards autoimmunity by inhibiting the function of CD4+ cells and not by depleting them. To determine if treatment with F(ab')2 anti-CD4 prevented lymphocytic infiltration of kidneys or simply inhibited the function of the infiltrating lymphocytes, long-term survivors of treatment with F(ab')2 anti-CD4 and intact anti-CD4 were sacrificed for immunohistochemical analysis of their kidneys. Untreated B/W mice had large lymphocytic aggregates under the surface epithelium of the renal calyces. Most of these lymphocytes were CD4+ T cells, but CD8+ T cells and B cells were also present. In contrast, treatment with either intact anti-CD4 or F(ab')2 anti-CD4 substantially reduced, and in many cases prevented, the development of renal infiltrates. Treatment with either form of anti-CD4 not only inhibited renal infiltration by CD4+ T cells, but also prevented the accumulation of CD8+ T cells and B cells. These observations suggest a role for the CD4+ T cell in the accumulation of lymphocytes in target organs.     

IgG on Human Blood Lymphocytes Studied by Immunofluorescence

Surface immunoglobulins (sIg) on human blood lymphocytes were identified by immunofluorescence (IFL) after staining with conjugated F(ab'2) fragments of the anti-Ig antibodies. A large fraction (approximately 20%) of freshly isolated lymphocytes was found to carry sIgG. Polyclocal IgG, which was present on Fc-receptor-bearing lymphocytes, could be removed by incubation and repeated washings only at 37 degrees C. Lymphocytes treated at 37 degrees C expressed the same percentage of sIg+ cells in direct IFL when F(ab')2 fragments of the antibody was used as when undigested aggregate-free IgG antibody was used. Indirect IFL using F(ab')2 fragments in both steps yielded similar sIg+ values. However, much higher percentage of cells carried sIg when undigested antibody was included in one of the steps. The results suggest that incubation and washing at 37 degrees C and the use of F(ab')2 fragments of the antibodies are important to eliminate absorbed sIgG and to avoid absorption of IgG during the staining procedure, thus preventing overestimation of the number of sIg+ B lymphocytes identified by IFL.     

LDH isoenzyme distribution in human T gamma lymphocytes

The lactate dehydrogenase (LDH) isoenzyme distribution was measured in T gamma+ lymphocytes from normal individuals. T gamma+ lymphocytes were obtained from purified T lymphocytes by ox-IgG rosette sedimentation. The B:A subunit ratio was clearly lower in the T gamma+ lymphocytes. Phenotyping of the T gamma+ lymphocytes showed a vast majority of OKT11+, OKM1+, OKT3- lymphocytes. In one experiment, T gamma+ lymphocytes were enriched by OKT3 depletion of T lymphocytes. The low B:A ratio was also found in these cells indicating that the LDH pattern is not the consequence of an in vitro activation by immune complexes. As the T gamma+ lymphocytes were considerably enriched in cells having the characteristics of NK cells according to their phenotyping, morphology and NK cell activity, we may assume that NK cells have a low B:A ratio.     

Promotion of natural killer cell growth in vitro by bispecific (anti-CD3 x anti-CD16) antibodies.

Bispecific heteroconjugated F(ab')2 fragments were prepared from pepsin-digested monoclonal OKT3 (anti-CD3) and 3G8 (anti-CD16) antibodies with 5,5'-dithiobis- (2-nitrobenzoic acid). When these bispecific antibodies (BsA) were added to peripheral blood lymphocyte (PBL) cultures with 100 U/ml human recombinant interleukin-2 (rIL-2), preferable growth of natural killer cells occurred. After 3 weeks the frequencies of CD56+ and CD56+3- cells in cultures with BsA were 74 +/- 7% and 65 +/- 7%, respectively, compared with 48 +/- 6% and 29 +/- 7% in control cultures. The frequencies of CD3+ lymphocytes in the presence of BsA, cells from 1-day cultures were labelled with fluorescein isothiocyanate (FITC)-conjugated anti-CD3, CD4 and CD8 monoclonal antibodies (mAb) and propidium iodide which stains dead cells. Flow cytometry revealed that more than 95% of the dead cells in cultures with BsA were CD3+. Thirty-seven per cent of CD3+, 43% of CD4+ and 17% of CD8+ cells were dead on day 1, and after 3 days the CD4+/CD8+ ratio among viable lymphocytes was 1.6 in the control and 0.5 in BsA cultures. Taken together, these results show that bispecific (anti-CD3 x anti-CD16) F(ab')2 fragments are strongly immunomodulatory by inducing the killing of T cells by CD16+ cells.     

Identification and isolation of OKT4+ suppressor cells with the monoclonal antibody WR16.

The monoclonal antibody WR16 was secreted by a hybridoma produced by fusing splenocytes from a BALB/c mouse immunized with human T-cell chronic lymphocytic leukaemia cells and the murine myeloma cell line NS-O. WR16 reacts specifically with human lymphocytes and binds to 48% of OKT4+ T lymphocytes and the majority of B lymphocytes. Human OKT4+ tonsil lymphocytes were subfractionated into WR16-/OKT4+ and WR16+/OKT4+ subpopulations by the panning technique. The capacity of these cells to help or suppress pokeweed mitogen-induced immunoglobulin (Ig) secretion by autologous B lymphocytes was monitored after 9 days of coculture. Cells of phenotype WR16-/OKT4+ enhanced Ig secretion in excess of that found with non-fractionated OKT4+ lymphocytes, whilst WR16+/OKT4+ lymphocytes suppressed Ig secretion when added to a mixture of B lymphocytes and non-fractionated OKT4+ cells. The WR16-/OKT4+ subpopulation was further fractionated by use of the MoAb Leu 8. Forty-two percent of WR16-/OKT4+ lymphocytes bound Leu 8, and cells of the phenotype WR16-/OKT4+/Leu 8+ were found to induce B-lymphocyte Ig secretion, whilst WR16-/OKT4+/Leu 8- lymphocytes were less active in this system. These data confirm the heterogeneity of the human T helper/inducer subset and indicate the existence of a population of OKT4+ lymphocytes that can suppress Ig secretion in the absence of OKT8+ lymphocytes.     

Heterogeneity of immunoregulatory T cells in human thyroid autoimmunity: influence of thyroid status.

Monoclonal antibodies of the OKT series were used to identify circulating T lymphocytes (OKT3+), their helper-inducer (OKT4+) and suppressor-cytotoxic (OKT8+) subsets and cells bearing Ia antigen (OKIa+) in 75 patients with thyroid autoimmune disorders, including 14 Graves' disease, 21 myxoedema, 20 asymptomatic thyroiditis, 12 Hashimoto's thyroiditis and eight simple goitre with superimposed thyroiditis. In the whole population of patients, a negative correlation was observed between the percentage of OKT8+ cells and serum free thyroxine levels whatever the type of thyroiditis. The percentage of OKT8+ cells was decreased in Graves' disease and increased in myxoedema while it reversed after adequate treatment of the two diseases. However, a trend to a decrease in the proportion of OKT8+ cells was still observed in treated Graves' disease and in all the other groups of thyroiditis with euthyroidism. The minor modifications observed for OKT3+ and OKT4+ cells were in relation with those of OKT8+ cells. There was an increased percentage of Ia+ cells in Graves' disease and in Hashimoto's thyroiditis partly reflecting the presence of activated lymphocytes. In conclusion, these data suggest first of all a direct influence of serum T4 on the distribution of circulating OKT8+ cells in addition to documenting the heterogeneity of T cell immunoregulatory factors.     

Unusually restricted anti-isotype human immune response to OKT3 monoclonal antibody

The unusual anti-mouse immunoglobulin (Ig) sensitization of a renal allograft recipient who was treated prophylactically with the anti-T cell monoclonal antibody OKT3 (IgG2a) is reported. Whereas in most patients, the injection of OKT3 (5 mg/day, i.v. for 13 days) induces the rapid appearance of neutralizing anti-OKT3 antibodies, the patient reported here did not show the signs of conventional anti-OKT3 sensitization. High levels of circulating OKT3 persisted and no OKT3+ lymphocytes reappeared during the whole treatment period. Moreover, no IgG or IgM anti-OKT3 antibodies were detected at any time, using a specific enzyme-linked immunosorbent assay. However, an atypical anti-isotype response was evidenced in this subject whose Ig were shown by indirect fluorescence to bind to normal T cells coated with OKT3 or with other anti-T cell murine monoclonal antibodies carrying the IgG2a isotype (no reactivity was observed with IgG1 or IgG2b molecules). The patient's Ig did not bind to normal T cells coated with F(ab')2 fragments of OKT3 and did not inhibit the binding of OKT3 to its target antigen indicating that they reacted with the Fc fragment of the OKT3 molecule. Additionally, and probably explained by this unusual anti-OKT3 response, the patient's Ig were shown to inhibit the phytohemagglutinin-induced proliferation of normal lymphocytes, to bind under selected in vitro conditions to normal T cells and lastly to enhance the antigenic modulation induced in vitro by OKT3 on its membrane receptor.     

Heterogeneity of concanavalin A-induced suppressor T cells in man defined with monoclonal antibodies.

Human peripheral blood T lymphocytes were enriched for OKT4+ or OKT8+ subpopulations using complement mediated lysis with OKT8 or OKT4 monoclonal antibodies. These subpopulations and unfractionated T cells were separately stimulated with concanavalin A (Con A) for a period of 48 hr and were then examined for their suppressive influence on proliferative response of autologous T cells to phytohaemagglutinin (PHA) or allogeneic non-T cells. Con A-activated unfractionated T cells, OKT4+ and OKT8+ T cell subsets markedly suppressed both these responses. Both OKT4+ and OKT8+ T cell subsets when enriched following Con A-activation of unfractionated T cells also caused significant suppression of proliferative responses of autologous T cells to PHA and allogeneic non-T cells in mixed lymphocyte cultures. The suppressive influence of Con A-activated T subsets was abolished by irradiation (2,000 rad) of activated cells. These studies indicate that Con A-induced suppressor T cells are heterogeneous. Precursors of Con A-induced suppressor T cells appear to reside in both OKT4+ and OKT8+ T cell populations.     

Relation between locomotion, chemotaxis and clustering of immune cells.

Experiments were designed to discover whether locomotor or chemotactic events are needed for clustering of lymphocytes with accessory cells or, conversely, whether clustering precedes the activation of lymphocyte locomotion. The time-courses of clustering and locomotor activation were compared and the behaviour of moving cells during cluster formation was filmed. Human lymphocytes direct from blood were activated by culture for 24-48 hr with anti-CD3 antibody or in allogeneic mixed leucocyte reactions (AMLR). The proportion of clustered and locomotor lymphocytes was low at the beginning of culture. Clusters appeared during the first few hours, before the increase in numbers of locomotor lymphocytes. Filming gave no evidence that the cells attracted one another chemotactically to form clusters. Rather, cells made chance contact by random locomotion and then remained adherent, though lymphocytes very close (less than or equal to 10 microns) to clusters did show increased pseudopod formation towards the cluster. However, the behaviour of motile lymphocytes responding to monocytes or macrophages given a phagocytic stimulus was different. Human monocytes which ingested opsonized zymosan released a material during but not following phagocytosis which caused an immediate increase in polar shape-change in lymphocytes. Macrophages from Corynebacterium parvum-induced mouse peritoneal exudates, given a phagocytic stimulus (opsonized Candida albicans), acted as sources of chemotactic gradients which attracted nearby lymphocytes to form clusters. This was due to brief release of a material immediately following phagocytosis, but after 15 min or so the macrophages no longer attracted nearby cells. These experiments suggest that, during induction of an immune response to a non-phagocytic stimulus, clusters form slowly by random contact followed by preferential adhesion. However, after phagocytosis, there may be a chemotactic response to the ingesting macrophage. This may help to focus lymphocytes onto macrophages which present microbial antigens.     

Endothelial cell lysis induced by lymphokine-activated human peripheral blood mononuclear cells

In vitro exposure of human peripheral blood mononuclear cells (PBMC) to interleukin 2 results in the generation of lymphokine-activated killer (LAK) cells. Such LAK cells exhibit cytotoxicity against a spectrum of tumor target cell lines whereas they apparently do not affect normal tissues. In this report we show that PBMC that have been activated with T cell growth factor lyse trypsinized human umbilical cord venous endothelial cells as well as endothelial cell monolayers in a dose-dependent manner. Microscopic analysis showed that during the 4-h incubation period cell clumps containing detached endothelial cells and LAK cells were formed. When these clumps were evaluated with trypan blue the endothelial cells stained positive whereas LAK cells excluded the dye. No lysis occurred when fresh PBMC were added to target endothelial cells. The endothelial cell kill could not be blocked with an anti-LFA-1 antibody nor with intact OKT3 or F(ab')2 fragments of WT32. We conclude that lymphokine-activated PBMC exhibit cell-mediated endothelial cell detachment and lysis.     

T-cell subsets in human lymphocytes maintained in IL-2 medium after PHA or mixed lymphocyte reaction activation

The culture of human T lymphocytes in interleukin-2 (IL-2) containing growth factor medium results in a significant shift in the T-lymphocytes subsets isolated from such cultures at weekly intervals. If normal peripheral blood mononuclear cells are stimulated with phytohemagglutinin (PHA) or in a mixed lymphocyte reaction (MLR), the resulting T lymphoblasts can be propagated in growth factor medium. Staining of the cultured cells with monoclonal antibodies was evaluated by indirect immunofluorescence on a laser-activated flow cytometer (Ortho Spectrum III). The antibodies used were: OKT3 (mature T lymphocytes), OKT4 (helper/inducer T lymphocytes), OKT8 (cytotoxic/suppressor T lymphocytes, OKT10 (immature and "activated" lymphocytes), OKT11a (cells which rosette with sheep erythrocytes), and OKIa-I (HLA-DR constant region). Both PHA and MLR activation resulted in initial preservation of the OKT4+ subset predominance over OKT8+ T lymphocytes noted on normal circulating blood lymphocytes. However, during culture in T-cell growth factor medium, there was a progressive increase in the percentage of OKT8+ cells, and a concomitant decrease in OKT4+ lymphoblasts. The increase in OKT8+ cells in the MLR-stimulated cultures was paralleled by an increase in specific cell-mediated cytotoxicity against the stimulating lymphocyte population. In addition to the shift in T-lymphocyte subset, there was virtual 100% staining with OKT3 and OKT11a, indicating the T-cell nature of the proliferating cells. OKT10 which was present on a small subset of fresh blood lymphocytes appeared rapidly in stimulated cultures, and was retained on virtually all lymphoblasts of either OKT4+ or OKT8+ subset. OKIa-1 cells increased slowly in PHA-stimulated cultures. HLA-DR+ T cells were detected earlier in MLR cultures. The activation of T lymphocytes results in a significant increase in the number of molecules of OKT11a bound per cell, in concert with the increased avidity of T lymphoblasts for sheep erythrocytes. The significant change in the phenotype and function of lymphoblasts isolated from long-term cultures demonstrates the importance of monitoring cultures, and the potential hazards in equating a cultured cell population with a freshly isolated one.     

OKT3 induces suppressor cells for mixed lymphocyte and PHA mitogenic responses in human peripheral lymphocytes

Human peripheral lymphocytes pretreated with the Orthoclone monoclonal anti- T cell antibody OKT3 for 48 h markedly suppressed the proliferative response of autologous lymphocytes in one-way MLC and the mitogenic response to PHA. The ability to induce suppression is specific to OKT3 since other monoclonal antibodies to human T cells (OKT1, OKT4 and OKT8) did not elicit similar responses. OKT3 is mitogenic but further proliferation of OKT3 pretreated lymphocytes was not required for the suppression of autologous lymphocytes since mitomycin-C treated cells were fully effective. Kinetic studies indicated that pretreatment of lymphocytes with OKT3 for 24 h was sufficient to induce marked inhibition of the mitogenic response of autologous lymphocytes to PHA whereas suppression in MLC was not observed until lymphocytes were pretreated for 48 h.These studies support the previous observations that OKT3 may be reaching with an important molecule on the T cell surface and that interaction of OKT3 with this molecule induces profound functional changes.     

Binding of monoclonal antibody to CD16 causes calcium mobilization in large granular lymphocytes but inhibits NK killing.

A monoclonal antibody (mAb), CLB/FcR gran I, reactive with the CD16 Fc receptor (FcRlo/FcRIII) of human cells, leads to calcium mobilization in large granular lymphocytes (LGL) but not in granulocytes. Identical responses are obtained with F(ab')2 fragments of this antibody, indicating that the response is independent of Fc-FcR binding, and that bivalent cross-linking of this receptor is adequate for optimal calcium mobilization. The calcium response was greater in CD3- LGL compared to CD3+ LGL, although the response was augmented in the latter cells by prior rosetting with sheep red blood cells (SRBC). Calcium mobilization in CD3- LGL induced by CLB/FcR gran I is associated with inhibition of natural killer cell (NK) killing, and inhibition of the enhanced NK killing induced by the anti-CD2 low-density monoclonal antibody, 9.1. This supports the view that the NK-enhancing activity of 9.1 is due to simultaneous binding to CD2 and CD16, and may in fact be transduced through the CD16 molecule. The variable reported effects of anti-CD16 antibodies on NK killing are likely to reflect the epitope bound rather than the isotype of antibody used, since F(ab')2 fragments of CLB/FcR gran I also inhibit NK killing.     

Separation and analysis of mononuclear cells infiltrating the thyroid of patients with Graves' disease

A simple method was established for separating lymphocytes infiltrating the thyroid from thyroid epithelial cells. Namely, suspensions of minced thyroid from patients with Graves' disease were layered on a Percoll two-step density gradient (p = 1.050 and 1.077 g/ml) and centrifuged (400g, 30 min, 4 degrees C). In this way 0.1-18 X 10(5) lymphocytes/g of thyroid tissue with a purity of 65-95% were obtained. Thyroid lymphocytes were analyzed quantitatively with monoclonal antibodies by laser flow cytometry and compared with peripheral lymphocytes. The proportion of OKT3+ cells was decreased with increase in OKIa+ cells. The percentage of OKIa+ cells was significantly correlated with that of Leu12+ cells. The percentages of OKT4+ cells and OKIa+ cells were higher when analyzed with an extended gate window, which was arranged for detection of activated, large-sized lymphocytes. The percentages of OKT8+ and Leu7+ cells were not significantly different from those in peripheral blood. From these results it was concluded that the proportion of B lymphocytes is increased and that of T lymphocytes is decreased, the proportion of activated B lymphocytes is increased, some helper/inducer T cells are activated in the thyroid gland in Graves' disease, and these activated lymphocytes may be important in local production of antithyroid autoantibodies.     

Enhancement of human B cell proliferation by an antibody to the C3d receptor, the gp 140 molecule

The C3d receptor is a specific marker of B lymphocytes. Recently we have shown that C3d receptor activity is carried by a gp 140 membrane antigen. A polyclonal antibody has been prepared by immunizing a rabbit with highly purified gp 140 molecule isolated from membranes of the human B lymphoblastoid cell line Raji and its high specificity was previously demonstrated. We tested the effect of this antibody to the C3d receptor on the B cell proliferative response. Purified B cells from human blood were induced to proliferate by a B cell growth factor (BCGF)-containing partially purified supernatant from activated T cells. The anti-C3d receptor F(ab')2 enhanced the BCGF-dependent B cell proliferation. This effect was dose dependent, was observed in the presence of different concentrations of BCGF and did not correspond to a change in the time course of the response. The anti-C3d receptor F(ab')2 had no mitogenic effect in the absence of T cell supernatant. In contrast the undigested anti-C3d receptor IgG suppressed the BCGF-dependent B cell proliferation. These results emphasize the potentialities of anti-gp 140 F(ab')2 to explore the involvement of the C3d receptor in the regulation of B cell response to T cell products.     

T cell activation by cross-linking anti-CD3 antibodies with second anti-T cell antibodies: dual antibody cross-linking mimics physical monocyte interaction

The anti-CD3 antibody BMA030 (IgG2a isotype) induces T cell activation and proliferation if an interaction with monocytes is provided. In contrast to other anti-CD3 antibodies, it is unable to induce interleukin (IL)2 responsiveness through cross-linking by plastic-bound goat anti mouse Ig antibodies (panning). Cross-linking BMA030 with a second anti-T cell antibody is, however, able to induce IL 2 responsiveness in monocyte-depleted T cell cultures. In this report we show that a large number of different antibodies are suitable for this dual antibody stimulation, and that the extent of proliferation corresponds to the percentage of T cells expressing the respective T cell antigen. Proliferation induced by low concentrations (0.1-1 ng/ml) of other anti-CD3 antibodies requires also cross-linking with second anti-T cell antibodies. The proliferative response of monocyte-depleted T cells to two cross-linked anti-T cell antibodies plus added IL 2 is of the same magnitude as the one induced by anti-CD3 antibodies plus monocytes. On the other hand, if monocytes are present, soluble anti-CD2, -CD4, -CD8, -LFA-1 antibodies (IgG1 or F(ab')2 fragments) can inhibit OKT3 or BMA030-induced T cell activation. Anti-CD6 antibodies do not interfere with this monocyte-dependent T cell stimulation. We conclude that dual antibody stimulation mimics the physical contact of T cells with monocyte membranes, where the T cell receptor CD3 complex is cross-linked with neighboring structures (mainly so-called adhesion molecules) through the interaction with respective counter-structures on monocyte membranes. Dual antibody cross-linking bypasses this interaction and can be used to stimulate IL 2 responsiveness of antibody-defined T cells.