Inhibitory effect of anti-class II antibodies on human B-cell activation

The role of class II antigens for B-cell activation was analyzed using purified human B cells and anti-class II monoclonal antibodies. The stimulation of purified B cells with Staphylococcus aureus Cowan I induced proliferation and differentiation into immunoglobulin-producing cells in the presence of interleukin-1 and T-cell-derived factors (B-cell growth factor and B-cell differentiation factor). The addition of anti-class II monoclonal antibodies inhibited B-cell responses. However, anti-class I monoclonal antibody did not inhibit B-cell responses. When mitomycin C and cycloheximide-treated B cells were added to the induction culture of B cells as the stimulator, B-cell responses were enhanced in a dose-dependent manner. Furthermore, the stimulator B cells also partially restored the suppressed B-cell responses which were induced by the pretreatment of B cells with anti-class II antibody. This enhancing effect of stimulator B cells on B-cell responses was inhibited by the pretreatment of stimulator B cells with anti-class II antibody. The treatment of B cells with anti-class II antibody and complement depleted the activity of both responder B cells and stimulator B cells. These results suggest that cellular interaction among B cells exists in the B-cell activation induced with Staphylococcus aureus, Cowan I and anti-class II antibody inhibits B-cell activation by interfering in this cellular interaction.     

Production of B cell-stimulating factors by B cells in patients with systemic lupus erythematosus

The production of B cell-stimulating factors (BSF) by B cells in patients with systemic lupus erythematosus (SLE) was studied in vitro. B cells from SLE patients markedly proliferated and differentiated into Ig-producing cells by in vitro culture without any stimulation. The culture supernatant of these B cells contained BSF activity that stimulated Staphylococcus aureus Cowan I-treated normal B cells to proliferate and differentiate into Ig-producing cells. By a Percoll gradient density centrifugation, BSF-producing cells were enriched in the higher density fraction, but were reduced in the lower density fraction. The BSF also stimulated the proliferation and the differentiation of SLE B cells. By a Percoll gradient density centrifugation, SLE B cells responsive to the BSF were enriched in the higher density fraction, but were reduced in the lower density fraction. The Mr of the BSF was estimated as about 18,000 Da by Sephacryl S-200 column chromatography. The BSF fraction did not possess IL-2 and IFN activity, but possessed IL-1 activity, which stimulated murine thymocyte proliferative responses. The BSF activity was partially, but not completely, absorbed by an anti-IL-1 alpha antibody. Furthermore, the BSF possessed IL-4 activity, which induced not only the proliferative responses of normal B cells stimulated with B cell mitogens, but also the expression of low affinity Fc epsilon R/CD23 on normal B cells. The BSF also possessed IL-6 activity, which induced the proliferative responses of IL-6-dependent hybridoma cells, MH-60 BSF2. Moreover, human rIL-1, rIL-4, and rIL-6 stimulated SLE B cells. These results suggest that SLE B cells spontaneously produce the BSF such as IL-1 alpha, IL-4, and IL-6 and express their receptors on their surface, and the interaction between the BSF and their receptors stimulates SLE B cells to spontaneously proliferate and differentiate into Ig-producing cells as an autocrine mechanism.     

Inhibitory effect of anti-class II antibody on the spontaneous activation of B cells in patients with systemic lupus erythematosus. Analysis with IL-1 production and IL-1 receptor expression

The mechanism of the spontaneous activation of B cells in patients with SLE was analyzed from the standpoint of the production of IL-1 from B cells and the expression of IL-1R on B cells. SLE B cells spontaneously produced IL-1-like factors which stimulated murine thymocyte proliferative responses. Their m.w. was about 17,000 and their isoelectric point was 4.8. The IL-1-like activity produced by B cells was absorbed with rabbit anti-IL-1 alpha antibody, but not with anti-IL-1 beta antibody. The differentiation of SLE B cells was enhanced by rIL-1 alpha, beta or IL-1-like factors produced by SLE B cells in a concentration-dependent manner. SLE B cells expressed large number of IL-1R detected by FITC-conjugated IL-1 alpha. By a Percoll gradient density centrifugation, IL-1-producing cells and B cells responsive to IL-1 were enriched in a higher density fraction, but were reduced in a lower density fraction. IL-1R-positive B cells were enriched in the lower density fraction, but were depleted in the higher density fraction. However, the expression of IL-1R on the lower density B cells was reduced by 2-day culture. The expression of IL-1R on the higher density B cells was increased during a 2-day culture. Anti-class II antibody inhibited the production of IL-1R on the higher density B cells. These results suggest that the cellular interaction among B precursor cells mediated by class II Ag induces the production of IL-1 and the expression of its receptors on their surface and the interaction between IL-1 and its receptors stimulates B precursor cells to spontaneously differentiate into Ig-producing cells as an autocrine mechanism in patients with SLE.     

Antibodies reactive with class II antigens encoded for by the major histocompatibility complex inhibit human B cell activation

Although class II antigens encoded by genes in the major histocompatibility complex (MHC) are important as recognition structures for immunoregulatory cell interactions, the precise functional role of these molecules in the biological responses of B lymphocytes is unknown. In the studies described here, we have examined the effects of six monoclonal antibodies reactive with human class II MHC antigens on B cell activation and proliferation. Peripheral blood IgM+ B cells purified by fluorescence-activated cell sorter (FACS) techniques were stimulated with anti-mu antibodies, protein A-bearing Staphylococcus aureus (SAC), or in T cell-dependent activation cultures. The B cell proliferative responses induced by these stimuli were inhibited 68 to 90% by low concentrations (1 to 5 micrograms/ml) of antibodies reactive with class II MHC antigens. Antibodies specific for DR and DQ antigens were both effective inhibitors of B cell proliferation. This inhibition was not due to the binding of antibody to B cell Fc-IgG receptors, because IgM and IgG anti-class II antibodies were equally potent as inhibitors. When responses of B cells fractionated on the basis of cell size by forward angle light scatter were analyzed, anti-DR and anti-DQ antibodies inhibited the proliferation of small, resting IgM+ cells induced by T-independent as well as T-dependent stimuli. Activation-dependent increases in B cell size and RNA synthesis were similarly inhibited. In contrast, the responses of large B cells (that had been preactivated in vivo) to T cell-derived B cell growth factors were not affected by anti-class II antibodies. These data suggest that class II MHC molecules do not serve merely as cellular interaction structures but also directly participate in early events of the B cell activation cascade that precede cell enlargement or increased RNA synthesis. After activation and expression of receptors for growth factors, however, B cell class II MHC antigens no longer mediate signals required for mitogenesis.     

B-B cell interactions in the spontaneous activation of B cells in autoimmune NZB mice.

We analyzed the mechanism of spontaneous B cell activation in lupus mice by using anticlass-II antibody in vitro. The in vitro culture of B cells from old NZB mice markedly produced Ig without any stimulation, while B cells from NZW mice did not. The addition of anticlass-II antibody (anti-Iad antibody) to the culture inhibited Ig production of NZB B cells in a concentration-dependent manner. On the other hand, the addition of anticlass-I antibody (anti-H-2Dd antibody) and anticlass-II antibody with different specificity (anti-Iak) gave no effect on the Ig production of NZB B cells. When mitomycin C-treated B cells were added to in vitro culture of responder B cells as a stimulator, Ig production of responder B cells was enhanced in a concentration-dependent manner. However, the enhancing effect of the stimulator B cells was abrogated by the pretreatment with anticlass-II antibody. The stimulator B-cell activity to NZB B cells was marked in NZB B cells, moderate in NZB/W F1 B cells, and weak in NZW B cells. Furthermore, the stimulator B-cell activity with regard to NZB B cells was marked in old female NZB B cells, moderate in old male NZB B cells, and weak in young NZB B cells. The expression of class II antigens on the surface of old female NZB B cells was significantly higher than that of old male NZB and young NZB B cells. These results suggest that in lupus mice the spontaneous B-cell activation is induced by an abnormal B-B cell interaction mediated by class II antigens.     

Effect of monoclonal antibodies (MoAb) to class I and class II HLA antigens on lectin- and MoAb OKT3-induced lymphocyte proliferation

We have examined the effect of several monoclonal antibodies (MoAb) to monomorphic determinants of class II HLA antigens, and MoAb to monomorphic determinants of class I HLA antigens and to beta-2-microglobulin (beta 2-mu) on lectin- and MoAb OKT3-induced proliferation of human peripheral blood mononuclear cells (PBMNC) and cultured T cells (CTC). Some, but not all, anti-class II HLA MoAb inhibited the proliferative response of PBMNC to MoAb OKT3 and pokeweed mitogen (PWM). The degree of inhibitory effect varied considerably. This effect was not limited to anti-class II HLA MoAb since anti-class I HLA MoAb and anti-beta 2-mu MoAb also inhibited MoAb OKT3- or PWM-induced proliferative responses. In contrast, the response of PBMNC to phytohemagglutinin (PHA) and concanavalin A (Con A) was not blocked by any anti-class II HLA MoAb. However, some anti-class II HLA MoAb also inhibited the proliferative response of CTC plus allogeneic peripheral blood adherent accessory cells (AC) to PHA or Con A as well as to MoAb OKT3 or PWM. This may be attributable to the substantially greater class II HLA antigen expression by CTC than by fresh lymphocytes. Pretreatment of either CTC or AC with anti-class II HLA MoAb inhibited OKT3-induced proliferation. In contrast, pretreatment of CTC, but not AC, with anti-class I HLA MoAb inhibited the proliferative response of CTC to OKT3. Pretreatment of CTC with anti-class I HLA MoAb inhibited PHA-, Con A and PWM-induced proliferation, to a greater degree than the anti-class II HLA MoAb. It appears as if lymphocyte activation by different mitogens exhibits variable requirements for the presence of cells expressing major histocompatibility determinants. Binding of Ab to membrane markers may interfere with lymphocyte-AC cooperation, perhaps by inhibiting binding of mitogens to their receptors or by interfering with lymphocyte and AC function. We also have examined the role of class II HLA antigens on CTC by depleting class II HLA-positive cells. As expected, elimination of class II HLA-positive AC with anti-class II HLA MoAb plus complement caused a decrease in proliferation of CTC in response to all the mitogens tested. In contrast, elimination of class II HLA-positive CTC was shown to clearly increase proliferation of CTC, perhaps because this may deplete class II HLA-positive suppressor cells.     

Induction of proliferation of human follicular (B type) lymphoma cells by cognate interaction with CD4+ T cell clones

We examined stimuli which are required for the induction of in vitro proliferation of follicular lymphoma cells, a low grade non-Hodgkin's B cell lymphoma characterized by a specific chromosomal translocation, t(14;18)(q32;q21), and by in vivo growth of the lymphoma cells in germinal center-like follicles infiltrated with CD4+ T cells. The purified follicular lymphoma cells, which are morphologically uniform, small, and dense, did not respond to stimulation with soluble lymphokines in the absence of T cells. Vigorous in vitro proliferation of follicular lymphoma cells was induced, however, when the follicular lymphoma cells were cultured with a CD4+ T cell clone which recognized alloantigens expressed by the lymphoma cells. This response required B-T cell contact, and was inhibited by anti-class II but not by anti-class I MHC mAb, indicating that these neoplastic B cells behaved as normal B cells and responded to normal activation and differentiation signals from T cells. After the cognate B lymphoma-T cell interaction occurred in culture, addition of IL-2 or IL-4 enhanced the proliferation of the tumor cells. These results, with a monoclonal and homogeneous population of B cells, affirm the idea that cognate interaction between B cells and Th cells is required for the effective activation of resting B cells. Moreover, these results suggest that a critical host-tumor interaction occurs in vivo, and that the polyclonal CD4+ T cells that infiltrate follicular lymphomas play a role in sustaining rather than inhibiting tumor growth in vivo. If so, therapies directed not only against the neoplastic cell but also against specific T cells and their cognate interactions with tumor cells may have a rationale.     

Immune regulation by monoclonal antibodies: failure to enhance mouse skin allografts

Monoclonal anti-H-2k alloantibodies were analyzed for their capacity to enhance the survival of B10.A skin grafted onto B10.D2 recipients. Included were five anti-class I and four anti-class II antibodies. In contrast to conventional B10.D2 anti-B10.A serum, none of the individual anti-class I or anti-class II monoclonal antibodies induced enhancement. The same negative results were obtained with various mixtures of anti-class I, anti-class II, or anti-class I + anti-class II antibodies. The failure to induce enhancement was not due to inefficient antigen binding in vivo, because monoclonal antibodies were as effective as conventional B10.D2 anti-B10.A serum in the induction of acute antibody-mediated graft rejection, and in the opsonization of 51Cr-labeled B10.A leukocytes injected into B10.D2 recipients pretreated with antibody. These results demonstrate that monoclonal antibodies cannot always substitute for conventional sera, at least not in immune regulation. They also show that although opsonization may be a prerequisite for the induction of enhancement, it does not guarantee that enhancement will invariably occur.     

Possible involvement of the T4 molecule in T cell recognition of class II HLA antigens: evidence from studies of proliferative responses to SB antigens

The T4 molecule has been identified as a marker of human T cell differentiation, but the function of this molecule remains to be defined. We have investigated its possible functional involvement in T cell proliferative responses to class II HLA antigens encoded by the recently described SB locus. The responses of SB-primed cells (specific for each of four different SB antigens) were studied with the use of two proliferation-inducing stimuli, SB antigen or TCGF. The proliferative responses to both stimuli were found to be mediated by T4+, T8- cells. Monoclonal antibodies against some epitopes on the T4 molecule (OKT4A and OKT4B) substantially blocked antigen-stimulated proliferative responses; antibodies against other epitopes of the T4 molecule (OKT4, T4C, T4D) blocked less well. Inhibition of SB-specific proliferation by antibodies to the T4 molecule was maximal only when the antibodies were incubated with the responder cells before the addition of stimulator cells. Proliferative responses of SB-primed cells stimulated with TCGF alone were not inhibited by any of the OKT4-related antibodies, but were completely inhibited by the anti-Tac monoclonal antibody, which reacts with the TCGF receptor. These results lend further support for the hypothesis that the T4 molecule is involved in T cell recognition of and/or activation by class II HLA antigens. We suggest that 1) the T4 molecule binds a nonpolymorphic epitope on class II HLA molecules, and 2) this interaction may facilitate, but not be an obligate requirement for, T cell activation by class II antigens.     

Analysis of T-T lymphocytes and T-accessory cell interactions using cloned T cells: MHC I restricted cloned T cells activate MHC II restricted T helper cells

We evaluated the role of molecules of the major histocompatibility complex (MHC) involved in the cellular interactions of two T-cell clones by testing the effect of monoclonal antibodies on the responses of the clones in vitro. The two T-cell clones used in the study are specific for minor histocompatibility antigens and restricted to the H-2K^k. In the absence of exogenous IL-2 the clones require the presence of Ia⁺, Thy-1⁻ accessory cells and of Thy-1⁺, Lyt-1⁺2⁻ cells in the irradiated spleen cell suspension used as stimulator. It is also necessary that both the accessory cells and the T cells in the stimulator cell populations are recognized specifically by the clones. Monoclonal antibodies specific for the H-2K product inhibited the lytic effector function of the cytolytic clone. These antibodies when added to cultures of stimulator cells and clones inhibited also the proliferation of this clone and of a nonlytic clone. When antigen recognition was measured by the increase in sensitivity of the clones to IL-2 while confronted with uv-irradiated stimulator cells, both clones were blocked efficiently by anti-H-2K antibodies. Thus, these results suggest that the interaction of monoclonal antibodies with the restricting H-2K molecule is sufficient to block the recognition signal, a prerequisite for proliferation. In contrast, monoclonal antibodies specific for A_αA_β and/or E_αE_β had no effect on cytolysis or on restricted recognition. However, they inhibited the proliferative responses as efficiently as the H-2K specific antibodies. Inhibition by class II-specific antibodies was not abolished when stimulator cell populations were depleted of Lyt-2⁺ cells. The blocking effect, however, was reversed by the addition of IL-2. No inhibition was obtained with antibody specific for E_αE_β when B10.A(4R) spleen cells, which do not express E_αE_β, or when B10.A(4R) accessory cells, which were reconstituted with (BALB/c X B10.A(4R)) F1 T cells, were used as stimulators. Stimulator cells heterozygous for H-2 could be inhibited by antibodies to the parental haplotype not encoded in the clones (H-2K^d). These and previous results suggest that H-2K-restricted minor histocompatibility antigen-specific recognition transmits an activating signal to the clones and to the stimulator cells. The clones probably are induced to express more IL-2 receptors. The stimulator T cells seem to interact through A_αA_β and E_αE_β molecules with syngeneic accessory cells. This interaction results in IL-2 production by the stimulator T cells and thus in the proliferation of the clones.     

Epstein-Barr virus induces normal B cell responses but defective suppressor T cell responses in patients with systemic lupus erythematosus

Epstein-Barr virus (EBV) is a polyclonal B cell activator, independent of helper T cells, which induces the generation of suppressor T cells in vivo and in vitro. Given the complexity of the immunologic abnormalities in patients with systemic lupus erythematosus (SLE), we used EBV as a tool to examine the following questions: a). Are SLE B cells primarily defective? and b). Does EBV stimulate the generation of suppressor activity from SLE T cells? It was found that B cells from SLE patients infected with EBV in vitro generate plaque-forming cell (PFC) responses that are similar to those raised by normal B cells infected with EBV within the first 14 days of culture. T cells from SLE patients, in contrast to T cells from normal individuals, cultured with autologous B cells plus EBV fail to develop the expected normal decrement of PFC during the late phase of the in vitro culture (day 14). However, B cells from SLE patients are susceptible to suppression as mixed cultures of SLE B cells and normal allogeneic T cells showed a pattern of PFC response to EBV similar to that of the co-culture of normal B cells with normal T cells. T cells from SLE patients, in analogous mixed cell cultures, failed to suppress either normal B cells or allogeneic SLE B cells. The above experiments indicate that the B cells are not intrinsically defective in SLE patients; rather, a specific T cell abnormality contributes to the lack of normal immunoregulation of certain B cell responses in SLE.     

Anti-tumor activity of class II MHC antigen-restricted cloned autoreactive T cells. I. Destruction of B16 melanoma cells mediated by bystander cytolysis in vitro

Two Lyt-1+, L3T4a+ autoreactive T cell clones specific for self-class II major histocompatibility complex (MHC) gene products were established from lymph node cells and spleen cells of C57BL/6J mice, respectively, by different methods. They were stimulated to proliferate in culture in response to I-Ab antigen-bearing syngeneic spleen cells in a class II MHC-restricted manner. This stimulation was inhibited completely by the addition of anti-L3T4a (GK1.5) or anti-I-Ab (3JP) monoclonal antibodies. The autoreactive T cell clones lysed syngeneic I-Ab+ target cells such as lipopolysaccharide (LPS) blasts. They also lysed I-A- bystander cells such as Cloudman and B16 melanoma and lymphoid tumor cells in the presence of I-Ab+ stimulator cells but not I-Ad+ cells. This bystander killing was most likely mediated by soluble factors released from the autoreactive T cells in response to I-Ab antigens, because culture supernatants from activated autoreactive T cells inhibited the proliferation of B16 melanoma cells in vitro and also had significant cytolytic activity. Both lymphotoxin and interferon-gamma were released from activated autoreactive T cells, suggesting that these cytotoxic lymphokines were responsible for autoreactive T cell-mediated cytolysis. The finding that the two clones, established independently and by different methods, show self-class II MHC antigen-restricted cytolysis, and bystander cytolysis suggests that these properties are not restricted to a unique population of autoreactive T cells. These results favor the concept that in vivo, autoreactive T cells may express not only regulatory activity in regard to antibody responses, but also anti-tumor activity via bystander cytolysis.     

Role of HLA class I and class II antigens in activation and differentiation of B cells

The monoclonal antibodies (MoAb) CR10-214, CR11-115, and Q1/28 to distinct monomorphic determinants of HLA class I antigens, the MoAb CL413 and PTF29.12 recognizing monomorphic determinants of HLA-DR antigens, the anti-HLA-DQw1 MoAb KS11, the anti-HLA-DPw1 MoAb B7/21, and the anti-HLA-DR,DP MoAb CR11-462 were tested for their ability to modulate human B-lymphocyte proliferation and maturation to IgM-forming cells. Purified tonsillar B cells were stimulated with Staphylococcus aureus bacteria of the Cowan first strain (SAC) or anti-human mu-chain xenoantibodies, as well as in growth factor- or T-cell-dependent activation cultures. The B-cell proliferative responses induced by SAC or by mitogenic concentrations of anti-mu-chain xenoantibodies were inhibited by some of the anti-HLA class I and anti-HLA class II monoclonal antibodies tested. The same antibodies were effective inhibitors of the proliferation of B cells stimulated with interferon-gamma (IFN-gamma) or interleukin-2 (IL-2) and with submitogenic concentrations of anti-mu-chain xenoantibodies. The proliferation induced by IL-2 of SAC-preactivated B cells was inhibited by some of the anti-HLA class II monoclonal antibodies, but not by the anti-HLA class I monoclonal antibodies tested. This inhibition appeared to reflect at least in part a direct effect on later events of the B-cell activation cascade, since some anti-HLA class II monoclonal antibodies still exerted considerable inhibitory activity when added together with IL-2 to SAC-preactivated B cells after the third day of culture. Anti HLA-DR, DQ, and DP monoclonal antibodies consistently inhibited the IgM production induced in B cells by T cells alone, T cells plus pokeweed mitogen (PWM), SAC plus IL-2, or IL-2 alone. In contrast, two of the three anti-HLA class I monoclonal antibodies tested inhibited the IgM production in cultures stimulated with SAC plus IL-2 and one the IgM production induced by IL-2 alone, but none of them had inhibitory effects on T-cell dependent IgM production. The results reported herein indicate that HLA class II molecules directly participate in different phases of the B-cell activation cascade. In addition, our data also suggest that HLA class I molecules can be involved in the events leading to B-cell proliferation and differentiation into immunoglobulin-secreting cells.     

Alloantigenic sites on class I major histocompatibility complex antigens: 61-69 region in the first domain of the H-2Kb molecule induces specific antibody and T cell responses

The most polymorphic residues in the first domain of class I major histocompatibility complex (MHC) molecules are in the 61-69 region. We have chosen the H-2Kb molecule for determining the role of this region in the induction of alloimmune responses. A synthetic peptide, Glu-Arg-Glu-Thr-Gln-Lys-Ala-Lys-Gly corresponding to this region was synthesized. T cells enriched from the lymph nodes of allostrain mice that were previously primed with H-2Kb containing cells or with the synthetic peptide in complete Freund's adjuvant undergo extensive in vitro proliferation in response to the synthetic (61-69)H-2Kb peptide. The response was dependent on the presentation of the (61-69)H-2Kb peptide by the syngeneic antigen-presenting cells and was blocked by anti-class II MHC monoclonal antibodies. This peptide fragment of class I MHC molecule activates only helper/inducer type T cells that are involved in the primary responses but not the effector cytotoxic T cells. When coupled to a carrier protein, (61-69)H-2Kb peptide induced antibodies in allostrain mice that bind to intact H-2Kb molecule. No antibodies or T cell responses could be induced in syngeneic H-2b mice. The antigenic site on the H-2Kb molecule recognized by two H-2Kb-specific monoclonal antibodies B8 X 3 X 24 and Y-25 was also mapped in the 61-69 region by direct binding to the synthetic peptide. Therefore the 61-69 region on the H-2Kb molecule represents the first defined sequence on a class I molecule that is directly involved in the induction of alloimmune responses.     

Anti-immunoglobulin stimulation of human B lymphocytes is inhibited by anti-class II major histocompatibility complex antibodies

The effect of murine monoclonal antibodies binding monomorphic epitopes of Class II, HLA-DR molecules on responding human B lymphocytes stimulated by anti-immunoglobulin M (IgM) antibodies was studied. Goat F(ab')2 anti-human IgM coupled to Sepharose beads (insoluble), or in solution, was added to macrophage-depleted B cells in culture with, or without, anti-human HLA-DR monoclonal antibodies. The addition of monoclonal anti-HLA-DR antibodies to anti-human IgM-stimulated B lymphocytes inhibited this T-independent B-cell proliferation by 82-94%. The role of Class II, HLA-DR molecules on B cells may therefore exceed that of antigen presentation alone, to include responding B-cell activation induced by anti-immunoglobulin.     

Receptor-like role of HLA-class I antigens: regulation of T cell activation

Class I major histocompatibility antigens are known to restrict the cytotoxic activity of T lymphocytes. However, experiments using monoclonal antibodies against class I antigens showed that these antigens also play some role in the regulation of T cell activation. Three monoclonal antibodies, namely W6/32 (anti-class I HLA-A, B, C, antigens), 4E (anti-class I HLA-B antigens), and BBM.1 (anti-beta 2-microglobulin) significantly suppressed the phytohemagglutinin-induced T cell proliferation. The inhibitory effect of anti-class I antibody was found to depend on the presence of monocyte/macrophage-type adherent cells. In the presence of antibody, adherent cells released a factor that suppressed T cell proliferation. These results suggest that HLA class I antigens on Mo1+ monocyte/macrophage cells function like ligand-receptor molecules, and regulate the secretion of suppressor factor(s).     

Modulation of human lymphocyte marker expression by gamma irradiation and mitomycin C.

gamma irradiation (GR) or mitomycin C (MC) treatment of stimulator cells is frequently used to achieve unidirectionality of response in the mixed lymphocyte reaction. As GR differs from MC in the pathways used to block lymphocyte replication, this study analyzes the effects of these modalities upon the expression of various differentiation and Class II major histocompatibility antigens on lymphocytes cultured for 24, 48, and 72 hr. There was a decrease in the mean density of HLA-DR expression on CD3+ and on CD8+ cells at 24, 48, and 72 hr after exposure to GR (42 and 35, 60 and 69, and 26 and 49%, respectively) or to MC (26 and 11, 26 and 18, and 46 and 30%, respectively). There was a parallel decrease in the levels of the corresponding cell subsets when compared with control cultured cells not exposed to GR or MC. In contrast, the density of HLA-DR markers on CD3-negative cells was increased at 24, 48, and 72 hr of culture following exposure to GR (73, 82, and 102%, respectively) or to MC (9, 45, and 80%, respectively). There was a more profound decrease in CD3+, CD8+, and CD19+ cell subset levels and in the density of the corresponding markers in GR-treated cells than in those of cells exposed to MC when the results were compared with those of untreated cultured control cells. Although GR appears to exert a more profound effect than MC, the results indicate that both modalities have the capacity to reduce the density of polymorphic determinants on Class II (HLA-D region)-encoded molecules on T (CD3+ and CD8+) and B (CD19+) cells which are known to trigger potent MLR responses. Both modalities may therefore affect profoundly the relative strength of MLC responses and the derived measurements of the degree of HLA Class II compatibility between stimulator and responder cells.     

Microinjection of macromolecules into normal murine lymphocytes by means of cell fusion. II. Enhancement and suppression of mitogenic responses by microinjection of monoclonal anti-cyclic AMP into B lymphocytes

Reproducible methods are now available for introducing protein molecules such as antibodies into normal murine lymphocytes by fusion with protein molecule-containing erythrocyte ghosts. Monoclonal antibodies against cyclic AMP were raised by hybridoma technique and packed into erythrocyte ghosts. Then, monoclonal anti-cyclic AMP containing ghosts were fused with splenic B lymphocytes by polyethylene glycol-mediated fusion at various intervals after LPS stimulation. This method made it possible for us to quantitatively microinject antibodies into B lymphocytes. Microinjection of anti-cyclic AMP antibody molecules into lymphocytes at a very early stage of LPS stimulation resulted in a marked enhancement of DNA synthetic responses as well as increased numbers of plaque-forming cells. Intracellular cyclic AMP levels were found to be markedly decreased after microinjection of monoclonal anti-cyclic AMP, suggesting that lowering the intracellular cyclic-AMP level in the B lymphocytes at an early stage of stimulation might have induced the enhanced proliferative as well as differentiative responses to LPS. Similar enhancing effects on cell proliferation were obtained when antibodies were injected 18 hr after stimulation. Microinjection of anti-cyclic AMP at 12 hr after culture, however, inhibited the DNA synthetic responses, and induction of plaque-forming cells was suppressed when anti-cyclic AMP was injected 6 hr after LPS stimulation. The present data suggest the biphasic regulatory roles of cyclic AMP at the early stage of B lymphocyte activation. This approach may be useful in identifying regulatory molecules in B lymphocyte induced by mitogenic or antigenic stimulation.     

Genetic restriction of autoreactive acetylcholine receptor-specific T lymphocytes in myasthenia gravis

Human autoreactive helper T lymphocytes with specificity for acetylcholine receptor (AChR) were isolated from three HLA-DR3-positive patients who had myasthenia gravis (MG), an autoimmune disease known to be associated with HLA-DR3 in the North European population. The antigen-specific T cells were evaluated for genetic restriction. Antigen presentation studies were performed with mitomycin C-treated accessory cells from a panel of HLA-typed unrelated donors. AChR-induced proliferation of the autoreactive T cells was maximal in the presence of autologous or HLA-DR-compatible antigen-presenting cells. In two DR-heterozygous patients both parental DR specificities served as restriction elements of the polyclonal AChR-reactive T cell populations. Preferential restriction to HLA-DR3 was observed in one patient, but this was also seen with PPD-specific T cells from the same donor. A series of monoclonal antibodies against HLA class II molecules was used for inhibition experiments. The inhibitory effects of the antibodies were not due to unspecific toxicity and could be observed after separate treatment of the antigen-presenting cells but not of the responding T cells. Several monoclonal antibodies against monomorphic HLA-DR determinants (DA 231, MAS 53, MAS 54, L243, OKIa1) had pronounced inhibitory effects. Anti-HLA-DQ(DC) monoclonal antibodies (Leu-10; TU 22) had only mild or no inhibitory effects in two patients but significantly inhibited AChR-specific T cells in one patient. A monoclonal antibody against HLA-DR3 (antibody 16.23) was not or was only weakly inhibitory in the DR3-positive patients, although it bound to autologous T line cells and B cells by indirect immunofluorescence. In one patient it was possible to compare the inhibition patterns of AChR-specific and PPD-specific T cells. Most of the monoclonal antibodies affected AChR- and PPD-specific T cells to a similar extent, but three antibodies (TU 22, 36, 39) inhibited PPD-specific T cells more than AChR-specific T cells, indicating the possibility of differential restriction of antigen- and autoantigen-specific T cells. It is suggested that the in vitro system described here may be helpful for the evaluation of anti-HLA class II antibodies as potential immunotherapeutic reagents.     

Role of class I and class II antigens in the allogenic stimulation: class I and class II recognition in allogenic stimulation; blocking of MLR by monoclonal antibodies and F(ab')2 fragments

Purified monomorphic monoclonal antibodies against Class I and Class II antigens in the inhibition of in vitro allogenic response were assayed. As expected, anti-Class II antibodies are highly inhibitory when used in concentrations greater than 5 micrograms/ml in MLRI and 50 micrograms/ml in MLRII. Surprisingly, anti-Class I monoclonal antibodies are as effective as anti-Class II in inhibiting primary MLR although they have no effect in MLRII. These results were confirmed by using F(ab')2 fragments. The inhibitory effect of anti-Class I has been shown to occur at the stimulator cell level. It is proposed that the allogenic stimulation is elicited after Class I and Class II recognition although only Class II differences are responsible for the proliferative response.