Staphylococcal Enterotoxin is a Bovine T Cell Superantigen

Different isoforms of soluble staphylococcal enterotoxins (SE): SEB, SEC-1 and SEC-2, were shown to stimulate bovine T cell proliferation, expression of cytokine messages, and IgG production. Intact metabolic function of APC was not required since peripheral blood mononuclear cells (PBMC), UV-irradiated prior to or following incubation with SE, were both capable of presenting SE, while PBMC treated with MAbs against MHC II lost the ability to stimulate T cell proliferation. SE caused approximately two fold increase of CD4⁺, and CD8⁺ T cells, but not MHC II⁺ APC or B cells. This model system suggests that SE transduces not only T cell activation signal, but also a non-proliferative signal for primary B cells to produce polyclonal IgG. We hypothesize that enterotoxin virulence may be in part due to its effect on activating the immune system.     

Staphylococcal Enterotoxin is a Bovine T Cell Superantigen

Different isoforms of soluble staphylococcal enterotoxins (SE): SEB, SEC-1 and SEC-2, were shown to stimulate bovine T cell proliferation, expression of cytokine messages, and IgG production. Intact metabolic function of APC was not required since peripheral blood mononuclear cells (PBMC), UV-irradiated prior to or following incubation with SE, were both capable of presenting SE, while PBMC treated with MAbs against MHC II lost the ability to stimulate T cell proliferation. SE caused approximately two fold increase of CD4⁺, and CD8⁺ T cells, but not MHC II⁺ APC or B cells. This model system suggests that SE transduces not only T cell activation signal, but also a non-proliferative signal for primary B cells to produce polyclonal IgG. We hypothesize that enterotoxin virulence may be in part due to its effect on activating the immune system.     

Bacterial lipopolysaccharide-induced immunoglobulin synthesis by human blood lymphocytes partially depleted of monocytes.

Bacterial lipopolysaccharide (LPS), a potent polyclonal B cell activator in rodents has not been found to be a consistent activator of human peripheral blood mononuclear cells (PBM). Since LPS activates monocytes to become suppressor cells, we asked whether depletion of monocytes would enhance the ability of LPS to induce in vitro activation and immunoglobulin synthesis and secretion by human B lymphocytes. Addition of 50 micrograms/ml LPS for 7 days to PBM cultures failed to induce a significant increase in IgM and IgG synthesis as measured by radioimmunoassay of culture supernatants. However, after partial depletion of adherent cells, the non-adherent cell population (NAC) produced large amounts of IgM and IgG (IgM: 696 ng/10(6) PBM vs 4236 ng/10(6) NAC, P less than 0.005; IgG: 68 ng/10(6) PBM vs 922 ng/10(6) NAC, P less than 0.02). The LPS-induced response was found to be T cell dependent and could be readily suppressed by the addition of autologous adherent cells. Addition of indomethacin to LPS-stimulated PBM did not result in increased Ig secretion. The poor response of human blood B cells to LPS may be due to the suppressive effect of activated monocytes.     

In vitro IgM and IgM rheumatoid factor production in response to Staphylococcus aureus Cowan I: evidence for the role of Leu-2-positive T suppressor cells and radiosensitive T helper cells.

Staphylococcus aureus Cowan I (SAC) is a potent stimulus of B cell proliferation and differentiation, the latter being T cell-dependent. It has been suggested that immunoglobulin and IgM rheumatoid factor (RF) production in response to SAC involves radiosensitive T helper cells. We studied normal peripheral blood mononuclear cell (PBMC) cultures to assess the roles of radiosensitive T cells and Leu-2 positive suppressor cells in the cellular control of SAC-stimulated IgM and IgM RF responses. Depletion of Leu-2 positive T cells from reconstituted autologous PBMC cultures resulted in an increase in SAC-stimulated IgM production in the majority of individuals, implying the involvement of Leu-2 positive suppressor T cells in this system. Suppression by Leu-2 positive cells is less evident in the SAC-induced IgM RF response, suggesting qualitative differences between IgM and IgM RF SAC-stimulated responses in PBMC cultures from the same normal individuals. Irradiation (1000 rads) of the T cell-enriched subpopulation, either with or without Leu-2 positive cell depletion, resulted in statistically significant decreases in IgM and IgM RF production in response to SAC in reconstituted autologous cultures, providing further evidence of a Leu-2 negative radiosensitive sheep-cell rosetting cell active in in vitro SAC responses. In contrast, PWM-stimulated PBMC cultures showed almost exclusively increases in total IgM and IgM RF production when T cells were irradiated (1000 rads) before culture, consistent with the radiosensitive T suppressor cell involved in the in vitro immunoglobulin responses to PWM. The same five out of nine individuals produced IgM RF, under different culture conditions, in response to PWM and SAC, suggesting that the ability of an individual to produce IgM RF lies intrinsically within the B cell.     

Effects of immunoglobulin G from patients with systemic lupus erythematosus on human B cell function

We examined the effect of systemic lupus erythematous (SLE) sera and Ig fractions on IgG and IgM release by cultured normal peripheral blood mononuclear cells (PBMC) when these cells were preincubated with serum dilutions or Ig fractions. Increases in both IgM and IgG (P less than 0.001 and less than 0.01) in cultured cell supernatants were recorded when PBMC were preincubated with SLE serum dilutions. IgG but not IgM from SLE was found to stimulate PBMC to release IgG (P less than 0.01). Similar results were obtained when SLE IgG was preincubated with adherent cell depleted cells (ADC) or isolated normal B cell fractions. When normal PBMC were preincubated with SLE serum or IgG and subsequently stimulated with pokeweed mitogen (PWM), a relatively blunted IgG release was observed (P less than 0.05); however, IgM release was significantly increased (P less than 0.001). This effect was not observed when PBMC were preincubated with SLE IgM, normal serum dilutions, or normal Ig fractions. Relative blunting of PWM response after PBMC were preincubated with SLE IgG was not reversed in PBMC depleted of adherent cells, OKT8+, or OKT9+ cells. Depletion of PBMC of LeuM1 cells increased IgG release in response to PWM when cells had been preincubated with SLE IgG. SLE serum or Ig fractions did not induce B cell growth factor release by T cells. SLE IgG appeared to act directly on B cell enriched populations to release IgG; this was not associated with significant increase in thymidine uptake, or apparent lysis of cells.     

The Stimulation of T Cells with Anti-CD2 Monoclonal Antibodies Facilitates the Induction of Polyclonal B-Cell Responses but does not Enhance the Activation of Antigen-Specific B Cells

In this report we compare the effect of stimulation of peripheral mononuclear cells (PBMC) by using two monoclonal antibodies (MoAb) directed against the CD2 receptor on T cells or by using autologous erythrocytes (E) which express on their surface lymphocyte function-associated antigen 3 (LFA3), a natural ligand for CD2. The addition of autologous erythrocytes to pokeweed mitogen (PWM)-stimulated PBMC results in the enhancement of polyclonal immunoglobulin synthesis and of antigen-specific B-cell responses. Because B cells lack the CD2 molecule, it can be concluded that their enhanced activity is a consequence of the delivery of activating signals by activated T lymphocytes. When PBMC cultures were stimulated with a pair of anti-CD2 MoAb (Leu5b and VIT13) we were able to induce polyclonal immunoglobulin synthesis, particularly IgM, in cultures supplemented with interleukin 2(IL-2). Specific responses to tetanus toxoid (TT) and keyhole limpet haemocyanin (KLH) were also enhanced by the addition of autologous E to PWM-stimulated PBMC. Significant anti-TT responses were observed in cultures stimulated with E + TT + IL-2. In contrast, stimulation of PBMC with VIT13 + Leu5b + IL-2 + antigen was not effective in inducing anti-TT antibody and only weakly effective in inducing anti-KLH antibodies. Replacing Leu5b by anti-CD3 had no effect on the induction of specific antibody responses; in contrast, replacement of Leu5b by E enhanced anti-TT antibody production while the effect on polyclonal production of IgM was minimal. Therefore, it appears that the signal delivered by the association of CD2 with LFA3 is a better potentiating signal for specific B-cell responses than the signal delivered by pairs of MoAb to different epitopes of CD2 or to CD2 and CD3 epitopes.     

Human T-cell responses to Mycoplasma arthritidis-derived superantigen.

When injected into mice, Mycoplasma arthritidis causes a chronic arthritis that resembles rheumatoid arthritis, histologically. The organism produces a superantigen termed Mycoplasma arthritidis mitogen or MAM, that in humans preferentially expands T cells whose antigen receptors express V beta 17. T cells with this phenotype appear to be increased in rheumatoid synovial effusions. We describe a novel approach to isolating and characterizing human MAM-reactive T-cell lines and determining their T-cell receptor (TCR) V beta usage. Lines were prepared from T cells that clustered with dendritic cells during a 2-day exposure to MAM. Cluster and noncluster fractions of T cells were then expanded by using feeder cells and a polyclonal mitogen. Most of the MAM reactivity was found in dendritic T-cell clusters, as were most of the T cells expressing TCR V beta 17. After expansion, 76% of the cluster-derived T-cell lines were MAM reactive, while no reactivity was seen in cell lines derived from the noncluster fraction. Of the MAM-reactive lines, 49% expressed V beta 17 on some or all of the cells. Cell lines from both cluster and noncluster fractions were analyzed for TCR V beta mRNA expression by PCR amplification. Other V beta genes (5.1, 7, 8, 12, and 20) were found to be expressed by lines that were MAM reactive, although these were not a major component of the cluster-derived T cells. Some non-cluster-derived lines expressed V beta s 17, 12, and 7, but these proved to be nonreactive to MAM. Therefore, dendritic cells can be used to immunoselect and characterize T cells that express superantigen-reactive TCRs.     

Clonal expansion of freshly isolated CD4T cells by randomized peptides and identification of peptide ligands using combinatorial peptide libraries

We synthesized Xn (n = 9 -- 19) peptides that consist of 9 to 19 residues with random sequences. X19 is considered to deliver antigenic stimuli to CD4 T cells, because: (a) X19 induces proliferation of peripheral blood mononuclear cells (PBMC), in the presence of IL-2, which is abrogated by monoclonal antibodies to class II HLA; (b) X19 + IL-2 induces proliferation of CD4 T cell clones of distinct specificities; and (c) T cell clones recognizing the same TCR ligands with distinct V beta usage are equally stimulated by X19 + IL-2. We next co-cultured single peripheral CD4 T cells with X19 and mitomycin-treated autologous PBMC. Indeed, single T cells of CD45RA(-) memory phenotype exhibited clonal expansion, with variable rates of proliferation, when IL-4, IL-7, IL-9, IL-15 and agonistic antibody to CD29 were included in the culture. These T cell clones showed heterogeneous proliferation patterns against KGXXXXXXXXXGK-based and KGXXXXXXXXXGKGKK-based combinatorial peptides libraries, in the presence of IL-2. Pattern-match search on a T cell clone resulted in peptide ligand candidates, one of which induced proliferation, as did protein molecules carrying the corresponding sequence. These results indicate that X19 can induce proliferation of peripheral memory T cells, the peptide ligands of which can be determined using combinatorial peptide libraries.     

Demonstration of a Helper Factor(s) with T-Cell-Replacing Activity in Synovial Fluid

Cell-free synovial fluid (SF) obtained from patients with rheumatoid arthritis contains a helper factor(s) capable of augmenting the generation of plaque-forming cells (PFC) in pokeweed mitogen (PWM)-stimulated normal peripheral blood mononuclear cells (PBMC). This helper factor behaves like a polyclonal B-cell activator, in that it triggers the formation of IgM, IgG, and IgA PFC. However, SF has little or no effect on the proliferation of PWM-activated PBMC. Furthermore, SF was capable of replacing T cells for PWM-induced differentiation but not proliferation of enriched human blood B lymphocytes. No helper factor or T-cell-replacing activity was found in SF from patients with traumatic synovitis. Fractionation of SF containing helper activity on staphylococcal protein A column indicated that the activity is induced by biologically active molecules distinct from materials that preferentially bind to protein A such as IgG immune complexes. We conclude that the present activity has striking similarities to the recently described B-cell differentiation factor that is produced by specifically activated T-cell lines in vitro.     

Skewed T cell receptor repertoire of Vdelta1(+) gammadelta T lymphocytes after human allogeneic haematopoietic stem cell transplantation and the potential role for Epstein-Barr virus-infected B cells in clonal restriction

The proliferation of Vdelta1(+) gammadelta T lymphocytes has been described in various infections including human immunodeficiency virus (HIV), cytomegalovirus (CMV) and malaria. However, the antigen specificity and functions of the human Vdelta1(+) T cells remain obscure. We sought to explore the biological role for this T cell subset by investigating the reconstitution of T cell receptor (TCR) repertoires of Vdelta1(+) gammadelta T lymphocytes after human allogeneic haematopoietic stem cell transplantation (HSCT). We observed skewed TCR repertoires of the Vdelta1(+) T cells in 27 of 44 post-transplant patients. Only one patient developed EBV-associated post-transplant lymphoproliferative disorder in the present patient cohort. The -WGI- amino acid motif was observed in CDR3 of clonally expanded Vdelta1(+) T cells in half the patients. A skew was also detected in certain healthy donors, and the Vdelta1(+) T cell clone derived from the donor mature T cell pool persisted in the recipient's blood even 10 years after transplant. This T cell clone expanded in vitro against stimulation with autologous EBV-lymphoblastoid cell lines (LCL), and the Vdelta1(+) T cell line expanded in vitro from the same patient showed cytotoxicity against autologous EBV-LCL. EBV-infected cells could also induce in vitro oligoclonal expansions of autologous Vdelta1(+) T cells from healthy EBV-seropositive individuals. These results suggest that human Vdelta1(+) T cells have a TCR repertoire against EBV-infected B cells and may play a role in protecting recipients of allogeneic HSCT from EBV-associated disease.     

Peptide length-dependent TCR antagonism on class II HLA-restricted responses of peripheral blood mononuclear cells and T cell clones

T cell clonal recognition of peptide ligands is highly diverse. To investigate how peptide mixtures with diverse sequences affect polyclonal responses of peripheral blood mononuclear cells (PBMC), we synthesized Xn (n = 9-19) peptides that consist of 9 to 19 residues with random sequences. We found that: (1) in antagonism assays, Xn peptides inhibit polyclonal responses of PBMC induced by purified protein derivative (PPD) and crude mite extracts as well as mixed lymphocyte reaction (MLR), in which intermediate-length peptides (n = 13 or 15) show the largest inhibitory effects; and (2) a high-affinity HLA-DR4-binding peptide is devoid of inhibitory activity against MLR to DR4, indicating that these effects are not caused by inhibition of peptide binding to HLA. Furthermore, X15 did not abrogate PBMC proliferation induced by phorbol 12-myristate 13-acetate + ionomycin, or anergize PBMC by preculture. All these observations indicate that TCR antagonism does exist at peripheral T cell levels in humans, and that Xn peptides, depending on peptide length, are capable of antagonizing T cell polyclonal responses. Indeed, even with cloned T cells, certain non-agonistic peptides shorter (but not too short) than the wild type in their C termini, function as TCR antagonists, findings which corroborate the observation that X13-15 antagonizes T cell responses more efficiently than does X17-19 or X9-11.     

Induction of T-cell proliferation by recombinant mouse and chimeric mouse/human anti-CD3 monoclonal antibodies.

The isotype of anti-CD3 mAb has a dramatic effect on anti-CD3 induced T-cell activation, as was previously reported for switch variants (IgG2b to IgA) of a high-avidity IgG1 anti-CD3 mAb (CLB-T3/4.1). In order to study and compare the isotype dependency of T-cell activation with anti-CD3 mAb of various mouse and human subclasses, we now prepared recombinant anti-CD3 mAb. The variable region of the anti-CD3 Ig heavy chain was cloned, joined with genes for the heavy chain constant region and expressed in a cell line only secreting autologous mouse chi light chains. Thus we obtained cell lines that produced mouse (m) IgM, mIgG3 and chimaeric mouse/human (h) IgM, hlgG1, hlgG2, hlgG3, hlgG4, hlgE and hlgA2 anti-CD3. The matched set of mouse and mouse/human chimaeric anti-CD3 isotypes switch variants was then used to study activation of T cells in an accessory cell-dependent system. hlgG1, hlgG4, hlgE, mlgG2a and mlgE induced T-cell proliferation in PBMC of all donors tested, whereas PBMC from a subset of donors were unresponsive to stimulation with hlgG2, hlgG3, hlgA2, mlgG1 and mlgG2b anti-CD3 mAb. hlgM, mlgM and mlgA were only able to induce T-cell mitogenesis in combination with PMA. Our panel of anti-CD3 mAb variants may prove a powerful tool to study mouse and human isotype-dependent effector functions and their influence on T-cell activation requirements in detail.     

Invariant natural killer T cells in lupus patients promote IgG and IgG autoantibody production

IgG autoantibodies, including antibodies to double‐stranded DNA (dsDNA), are pathogenic in systemic lupus erythematosus (SLE), but the mechanisms controlling their production are not understood. To assess the role of invariant natural killer T (iNKT) cells in this process, we studied 44 lupus patients. We took advantage of the propensity of PBMCs from patients with active disease to spontaneously secrete IgG in vitro. Despite the rarity of iNKT cells in lupus blood (0.002–0.05% of CD3‐positive T cells), antibody blockade of the conserved iNKT TCR or its ligand, CD1d, or selective depletion of iNKT cells, inhibited spontaneous secretion of total IgG and anti‐dsDNA IgG by lupus PBMCs. Addition of anti‐iNKT or anti‐CD1d antibody to PBMC cultures also reduced the frequency of plasma cells, suggesting that lupus iNKT cells induce B‐cell maturation. Like fresh iNKT cells, expanded iNKT‐cell lines from lupus patients, but not healthy subjects, induced autologous B cells to secrete antibodies, including IgG anti‐dsDNA. This activity was inhibited by anti‐CD40L antibody, as well as anti‐CD1d antibody, confirming a role for CD40L‐CD40 and TCR‐CD1d interactions in lupus iNKT‐cell‐mediated help. These results reveal a critical role for iNKT cells in B‐cell maturation and autoantibody production in patients with lupus.     

Down-regulation of antigen-specific antibody production by TCR antagonist peptides in vivo

The efficacy of TCR antagonist peptides in inhibition of antigen-specific antibody production and T cell responses in vivo was evaluated. Among amino acid-substituted analogs of a peptide corresponding to residues 119 - 133 of bovine beta-lactoglobulin (p119 - 133), pR124Q and pD129S, prepared by substitution of Gln and Ser for Arg(124) and Asp(129), respectively, have been shown to display TCR antagonist activity for three out of four distinct p119 - 133-specific T cell clones and for polyclonal T cells derived from p119 - 133-immunized C57BL / 6 mice. Both pD129S and pR124Q inhibited in vivo priming and subsequent activation of T cells by p119 - 133 when co-injected with p119 - 133 into mice, as shown by the decreased proliferation of T cells in response to p119-133 in vitro. pD129S significantly inhibited production of anti-p119 - 113 antibodies of IgG1, IgG2b and IgE isotype in vivo when co-injected into mice together with p119 - 133 at the time of the first immunization. However, pR124Q was totally ineffective in inhibition of the antibody responses. Anti-p119 - 133 antibodies from p119 - 133-immunized mice could bind to pR124Q but not to pD129S, suggesting that the difference in cross-reactivity is responsible for the different effect of these two peptides on specific antibody production. Our findings demonstrate that a single TCR antagonist peptide can inhibit antigen-specific polyclonal antibody production when this antagonist peptide does not cross-react with the antibody elicited in response to an antigenic peptide.     

Extensive CD4 cross-linking inhibits T cell activation by anti-receptor antibody but not by antigen.

Anti-CD4 mAbs have been shown to inhibit T cell activation in a variety of ways. We have tested a panel of IgG and IgM anti-CD4 mAbs for their effects on the activation of a cloned T cell line by antigen presented by syngeneic accessory cells, by soluble anti-T cell receptor antibodies, and by mitogenic lectins. Both IgM and IgG mAbs to CD4 inhibit responses to mitogenic lectins. However, IgM, but not IgG, anti-CD4 antibodies inhibit T cell activation by mAbs specific for the TCR. This inhibitory activity appears to be due to the signaling effects of IgM mAbs, as cross-linked IgG antibodies mimic the behavior of the IgM anti-CD4 antibodies. Inhibition of T cell activation correlates with the ability of IgM and of cross-linked IgG anti-CD4 antibodies to induce tyrosine phosphorylation of the CD4-associated tyrosine kinase p56lck and an unknown substrate, pp32. Surprisingly, we find that the IgM anti-CD4 mAbs tested had no effect on the specific antigen recognition, despite their potent inhibitory effects on the other responses of the same cloned T cell line. These results suggest that multivalent CD4 interactions with ligands such as MHC class II molecules are inhibitory of T cell activation, but that this inhibition can be reversed when CD4 and the TCR bind the same ligand. We discuss the possible implications for positive intrathymic selection of these findings on signaling through CD4.     

Pokeweed mitogen-induced immunoglobulin secretory responses of thymic B cells in myasthenia gravis: selective secretion of IgG versus IgM cannot be explained by helper functions of thymic T cells

The thymus, with its striking B cell infiltrates, is widely regarded as an important element in the pathogenesis of myasthenia gravis (MG) but its role remains to be elucidated. To gain further insight into the functional properties of MG thymic B cells, we studied the heavy chain isotype of immunoglobulin they produced in vitro in response to the T cell-dependent polyclonal activator pokeweed mitogen (PWM). MG thymic cells secreted prominent amounts of IgG but little IgM. In contrast, peripheral blood mononuclear cells (PBM) of the same subjects secreted similar amounts of IgG and IgM as did PBM of control subjects. In cell admixture experiments, MG thymic T cells, like PBM T cells, helped autologous PBM B cells produce IgM as well as IgG, although the overall magnitude of help for both isotypes appeared less than that of PBM T cells. Thus, in response to PWM, MG thymic B cells are largely committed to an IgG response and this likely reflects the intrinsic properties of these cells rather than the immunoregulatory properties of thymic T cells. This IgG isotype switch likely reflects in vivo activation events.     

CD4 T lymphocyte activation in BLV-induced persistent B lymphocytosis in cattle

Bovine leukemia virus (BLV) is an oncogenic retrovirus in the human T cell leukemia virus family. BLV infects B lymphocytes and induces a nonmalignant persistent lymphocytosis (PL) and leukemia/lymphoma in cattle. There is evidence that CD4 T lymphocytes are activated during BLV infection and promote the development of PL. How CD4 T lymphocytes are activated by BLV infection is not known. We observed that CD4 T lymphocytes from PL cattle proliferated in the presence of autologous, irradiated peripheral blood mononuclear cells (PBMC), whereas no proliferation occurred in cell cultures from BLV-infected non-PL cattle. Proliferation required direct contact with metabolically active irradiated PBMC but was not associated with viral protein expression or inhibited by antibodies to BLV. Unexpectedly, B lymphocytes alone failed to account for the irradiated PBMC stimulation of CD4 T lymphocytes. These observations and the magnitude of the proliferative response suggest that activation is polyclonal and involves mechanisms other than BLV antigen-specific stimulation.     

Testosterone inhibits immunoglobulin production by human peripheral blood mononuclear cells

We studied the in vitro effect of testosterone on spontaneous immunoglobulin production by human peripheral blood mononuclear cells (PBMC). Testosterone inhibited IgG and IgM production by PBMC both from males and females. The inhibitory effect of testosterone was revealed at doses more than 1 nm, increased dose-dependently, and reached a plateau at 100 nm. At doses <1000 nm, testosterone did not reduce cell viability. Testosterone treatment reduced IgG production by 59.0% and that of IgM by 61.3% compared with control. Immunoglobulin production by B cells was also suppressed by testosterone, though the magnitude of the suppressive effect on B cells was lower than that on whole PBMC; testosterone-induced decrease of IgG production compared with control was 26.9% and that of IgM was 24.9%. Exogenous IL-6 partially restored the impaired immunoglobulin production of testosterone-treated PBMC; IgG production in testosterone culture was increased by IL-6 from 35.6% to 66.5% of control and that of IgM was also increased from 38.9% to 71.2%, respectively. Testosterone treatment reduced IL-6 production of monocytes by 78.4% compared with control, but neither affected that of T cells or B cells. These results suggest that testosterone may suppress immunoglobulin production of human PBMC directly by inhibiting B cell activity and indirectly by reducing IL-6 production of monocytes. It is thus indicated that this hormone may have protective and therapeutic effects on human autoimmune diseases.     

Induction of T cell proliferation with anti-CD3 switch-variant monoclonal antibodies: effects of heavy chain isotype in monocyte-dependent systems

Monoclonal antibodies (mAb) directed against the CD3 (T3), antigen are able to induce proliferation in resting human T lymphocytes. T cell proliferation only occurs in the presence of monocytes that carry the proper Fc receptor for the mAb used. To further analyze the role of the Fc portion of anti-CD3 mAb in proliferation induction, we isolated, starting from a gamma 1 anti-CD3-producing hybridoma, four heavy-chain isotype switch-variant antibody-secreting clones, producing gamma 2b, gamma 2a, epsilon and alpha, respectively. All variant antibodies recognize the CD3 antigen as determined by immunoprecipitation and cross-blocking experiments. With this series of isotype variant antibodies we were able, in proliferation induction experiments, to confirm the Fc receptor polymorphism for murine IgG2a, IgG2b and IgG1 on human monocytes. Moreover, we found that all 30 donors tested responded to the IgE anti-CD3 antibody, while no IgA responders could be identified. The induction of proliferation by the IgE variant antibody does not require the 72-kDa Fc receptor which is responsible for the interaction with mouse IgG2a. Nonresponsiveness to the IgG1 antibody, but not to the IgG2b or IgA variant antibodies, could be overcome by the addition of exogenous interleukin 2 to the cultures. When the switch-variant antibodies were used to induce IgM synthesis in peripheral blood mononuclear cells only low IgM synthesis was found, with the exception of the IgE variant, which induced excellent T cell help for IgM production.     

Human CD4-reactive antibodies from sle patients induce reversible inhibition of polyclonal t lymphocyte proliferation

We report on isolation of human polyclonal CD4-reactive antibodies of IgM and/or IgG isotypes from several SLE patients. These antibodies bound specifically to CD4-expressing cell lines and to rCD4 in ELISA and immunoblots. Saturation of CD4-binding sites occurred at antibody concentrations between 5 and 15 μg/ml. Anti-CD4 antibodies, in a dose-dependent manner, suppressed the proliferative responses of human peripheral blood mononuclear cells (PBMC) to superantigens (Staphylococcal enterotoxins A and B), anti-CD3 antibodies, and mitogens (PWM and Con A, but not PHA). They could also inhibit the proliferation of highly purified human T cells induced by immobilized anti-CD3 antibodies. To promote their effects on T cells, human antiCD4 antibodies had to be present at lymphocyte cultures before or at the time of priming. There was no significant inhibition when antibodies were added more than 24 h following T cell activation. Substantial evidence that the immunosuppression induced by anti-CD4 antibodies was due to their direct effect on T cells was obtained. Downregulatory effect of anti-CD4 antibodies could be significantly reversed by addition of exogenous IL-2 and by preincubation with soluble recombinant (r)CD4. Interestingly, at least one affinity-purified anti-CD4 antibody could costimulate the T cell proliferation induced by superantigens or anti-CD3 antibodies, especially when used at subsaturating concentrations (1–4 μg/ml) and when added subsequently to the initiation of cultures.