Inhibition of Anti-CD3 and Interleukin-2 Stimulated T Lymphocyte Proliferation by Peptidomimetic Opioid Compound

In continuation to our earlier studies with peptidomimetic opioid compounds, we have further investigated immunosuppressive properties of one of our peptidomimetic compound (Tyr-NH-CH₂-CH₂-O-Phe-NH₂) using peripheral blood mononuclear cells (PBMCs) of healthy volunteers. Peptidomimetic compound was evaluated for its effect on anti-CD3 and recombinant human interleukin-2 (rhIL-2) stimulated lymphocyte proliferation in vitro and lipopolysaccharide (LPS) induced activation of mitogen activated protein kinase (MAPK, pp42/44) in mouse macrophage cells (RAW 264.7). Our results show the immunosuppressive potential of synthetic peptidomimetic compound. This compound significantly inhibited anti-CD3 and rhIL-2 stimulated lymphocyte proliferation in vitro. However, this peptidomimetic compound did not show any effect on LPS induced MAPK activation. These observations suggest that above peptidomimetic compound has potential to inhibit immune responses mediated by lymphocytes.     

A new carboxamide compound exerts immuno-suppressive activity by inhibiting dendritic cell maturation

The immunosuppressive properties of a benzamide derivative, JM34, previously characterized as an anti-inflammatory compound are described. The immunosuppressive potential of JM34 was evidenced by inhibition of PBMC proliferation in vitro with an IC50 of 20 microM. In contrast with classical immunosuppressive drugs, JM34 affected neither cytokine production nor IL-2R expression from activated T cell clones, and displayed only moderate inhibition of IL-2-induced or anti-CD3/anti-CD28-induced proliferation. We investigated its effects on dendritic cells (DC) in vitro. Addition of JM34 during DC maturation inhibited the expression of some maturation markers: specifically, MHC molecule up-regulation was totally inhibited and CD83 expression was significantly reduced, while up-regulation of CD86, CD80 or CD40 was less affected. Moreover, JM34-treated DC showed impaired IL-12 but not IL-10 secretion, and a markedly reduced ability to present antigens to naive T lymphocytes in vitro. We provide evidence that these JM34-induced alterations of DC were associated with a marked inhibition of NF-kappaB nuclear translocation. Finally, JM34 inhibited delayed type hypersensitivity dose dependently in mice. In conclusion, our data suggest that JM34 inhibited T lymphocyte activation mainly by targeting DC, and thus may represent a new class of therapeutic agents in the fields of transplantation and autoimmune diseases.     

Interleukin-10-induced CD8 cell proliferation

Interleukin (IL)-10, a product of T helper 2 (Th2) lymphocytes, has been shown to be an important regulator of lymphoid and myeloid cells, inhibiting mitogen, peptide and alloantigen-induced T-cell proliferation and IL-2 production. The microenvironment at the time of cell activation, notably the presence or absence of cytokines such as IL-10, interferon-gamma (IFN-gamma) and IL-2, is believed to determine the lineage and magnitude of cell-mediated responses. In this study, we show that recombinant human IL-10 (rhIL-10) exerts a dose-dependent inhibitory effect on human peripheral blood mononuclear cells stimulated in vitro, when these cells have not previously been exposed to rhIL-10. Furthermore, incubation of these cells with high doses of rhIL-10, either before or at the time of activation, results in inhibition which is followed several days later by the emergence of a population of CD8 positive cells. This rhIL-10-responsive CD8, positive cell population still emerges even when the cells are washed following incubation with rhIL-10 prior to cell activation. Using purified CD8 populations this was shown to be a direct action of rhIL-10 on CD8 cells and not via CD4 positive cells and monocytes. This finding was only observed when cells were activated with a cross-linking anti-CD3 antibody and not when activated with phorbol-12-mystrate-13-acetate (PMA) and calcium ionophore (CaIon), suggesting that the effect is mediated through cell-surface receptors. Analysis of CD8 positive clones reveal production of Tc2 patterns of cytokines and reduced cell cytotoxicity to allogeneic, natural killer and lymphokine activated cell targets.     

Monoclonal antibodies to B and T lymphocyte attenuator (BTLA) have no effect on in vitro B cell proliferation and act to inhibit in vitro T cell proliferation when presented in a cis, but not trans, format relative to the activating stimulus

B and T lymphocyte attenuator (BTLA) is an immunoglobulin superfamily member surface protein expressed on B and T cells. Its ligand, herpesvirus entry mediator (HVEM), is believed to act as a monomeric agonist that signals via the CRD1 of HVEM to inhibit lymphocyte activation: HVEM is also the receptor for lymphotoxin-α and LIGHT, which both bind in the CRD2 and CRD3 domains of the HVEM molecule, and for CD160 which competes with BTLA. We have shown that recombinant HVEM and a panel of different monoclonal antibodies specifically bind murine BTLA on both B and T cells and that some antibodies inhibit anti-CD3ε-induced T cell proliferation in vitro, but only when constrained appropriately with a putatively cross-linking reagent. The antibodies had no significant effect on in vitro T cell proliferation in a mixed lymphocyte reaction (MLR) assay nor on in vitro DO11.10 antigen-induced T cell proliferation. None of these antibodies, nor HVEM-Fc, had any significant effect on in vitro B cell proliferation induced by anti-immunoglobulin M antibodies (±anti-CD40) or lipopolysaccharide. We further elucidated the requirements for inhibition of in vitro T cell proliferation using a beads-based system to demonstrate that the antibodies that inhibited T cell proliferation in vitro were required to be presented to the T cell in a cis, and not trans, format relative to the anti-CD3ε stimulus. We also found that antibodies that inhibited T cell proliferation in vitro had no significant effect on the antibody captured interleukin-2 associated with the in vivo activation of DO11.10 T cells transferred to syngeneic recipient BALB/c mice. These data suggest that there may be specific structural requirements for the BTLA molecule to exert its effect on lymphocyte activation and proliferation.     

Phenotypic Convergence and Divergence of Surface Immunoglobulin and CD40 Signals

Both anti-CD40 antibodies and anti-immunoglobulin (Ig) coupled to Sepharose induced proliferation of resting B cells and suppressed lipopolysaccharide (LPS)-induced B-cell differentiation to immunoglobulin secretion at comparable levels determined with the plaque-forming assay and Ig RNA steady state levels. Anti-CD40 antibodies also increased the proliferation of B cells stimulated by T helper cells in vitro while suppressing their differentiation to Ig secretion. Further, B cells preactivated by anti-Ig, anti-CD40 or a combination of the two mitogens could be restimulated by anti-CD40 but not by anti-Ig antibodies. Phenotypic divergence of Ig and CD40 signals regarding surface expression of activation markers was observed. Restimulation of anti-Ig- or anti-CD40-prestimulated cells with anti-Ig induced apoptosis whereas apoptosis could be inhibited when cells were recultivated with anti-CD40.     

Trypanosoma cruzi induces regulatory dendritic cells in vitro

A main feature of acute infection with Trypanosoma cruzi is the presence of immunological disorders. A previous study demonstrated that acute infection with the virulent RA strain downregulates the expression of major histocompatibility complex class II (MHC-II) on antigen-presenting cells and impairs the T-cell stimulatory capacity of splenic dendritic cells (DC). In the present work, we assessed the ability of trypomastigotes (Tp) to modulate the differentiation stage and functionality of bone marrow-derived DC in vitro. We observed that the Tp stage of T. cruzi failed to activate DC, which preserved their low expression of MHC-II and costimulatory molecules, as well as their endocytic activity. We also show that Tp induced transforming growth factor beta (TGF-beta) secretion by DC and enhanced the gap between interleukin-10 (IL-10) and IL-12p70 production, showing a higher IL-10/IL-12p70 ratio upon lipopolysaccharide (LPS) treatment. In addition, we observed that Tp prevented DC full activation induced by LPS, thereby downregulating their MHC-II surface expression and inhibiting their capacity to stimulate lymphocyte proliferation. In vitro IL-10 neutralization during the differentiation process of DC with Tp+LPS showed a reversion of their inhibitory effect during mixed lymphocyte reaction. In contrast, only simultaneous neutralization of IL-10 and TGF-beta, after DC differentiation, was involved in the partial restitution of lymphocyte proliferation. Since both TGF-beta and IL-10 are immunosuppressive cytokines essential in the modulation of the immune response and important in the induction of tolerance, our results suggest for the first time that Tp are responsible for the generation of regulatory DC in vitro.     

Bacterial lipopolysaccharide induced B cell activation is mediated via a phosphatidylinositol 3-kinase dependent signaling pathway.

Bacterial lipopolysaccharide (LPS) is a potent stimulant of B cells and macrophages. LPS induces B cell proliferation and differentiation into antibody secreting cells. In addition, LPS also stimulates IL-6 secretion in mature B cells and in immature B cell lines such as WEHI-231. Although sufficient literature is available on LPS induced signaling events in monocytes and macrophages, the mechanisms involved in LPS induced B cell activation are not well understood. In this report, it is shown that both LPS mediated B cell proliferation and IL-6 secretion are dependent on phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathways. The B cell specific co-receptor, CD19 is not tyrosine phosphorylated in LPS stimulated B cells. Thus, in contrast to B cell antigen receptor (BCR) signaling, the activation of PI 3-kinase appears not to be related to the recruitment of PI 3-kinase to tyrosine phosphorylated CD19. This is the first demonstration of the importance of PI 3-kinase signaling pathway in LPS mediated B lymphocyte activation.     

人白细胞介素-21 cDNA克隆及其在大肠杆菌系统的表达

目的：克隆人细胞因子IL-2l全长cDNA及其在大肠杆菌中表达，并进一步检测其表达产物的功能。方法：抽取外周血，分离淋巴细胞；抗CD3抗体刺激后，提取总RNA，通过RT-PCR获得两个部分重叠的IL-21 cDNA片段(分别为5′端242bp，3′端425bp)，重组PCR扩增出IL-21全长cDNA：DNA测序正确后，将成熟IL-21 cDNA的编码框序列构建到原核表达载体pET28a( )中。卡那霉素平板筛选及PCR鉴定，挑出阳性克隆进行扩增、转化大肠杆菌进行IPTG诱导表达：SDS—PAGE、Western blotting鉴定，亲和层析分离纯化得到M，为18600的带有组氨酸标签重组人IL-21融合蛋白。透析法重折叠复性，MTT法测定其对T细胞增殖活性。结果：成功获得人IL-21全长cDNA，并以包涵体形式表达于大肠杆菌中。重折叠后的rhIL-21融合蛋白具有与抗CD3抗体共刺激T细胞增殖作用。结论：获得具有生物学活性的rhIL-21细胞因子，为进一步研究其功能奠定基础。     

Immobilized anti-CD3 monoclonal antibodies induce accessory cell-independent lymphokine production, proliferation and helper activity in human T lymphocytes.

Monoclonal antibodies (mAb) directed against the human CD3 molecular complex are able, when immobilized on the plastic of microtitre wells, to induce accessory cell-independent T-cell proliferation. In this study, we show that the anti-CD3 mAb CLB-T3/3 induces strong T-cell stimulation that is proportional to the density of the immobilized antibody. T cells, optimally stimulated with plastic-immobilized CLB-T3/3, showed a five-fold higher proliferation compared to cells that were stimulated with soluble anti-CD3 in the presence of accessory cells. The difference in magnitude of proliferation was found to be correlated with the expression of the CD25 (TAC) antigen and the production of interleukin (IL)-2, but not with the number of high-avidity IL-2 receptors expressed on the surface of these differentially activated cells. In addition, immobilized CLB-T3/3 initiated the production of interferon-gamma (IFN-gamma), but not of IL-4, in purified T lymphocytes. Coated anti-CD3 mAb induced helper activity in T cells for IgM and IgG production by B lymphocytes. Whereas addition of IL-1 or IL-2 had only a moderate effect on T-cell proliferation induced by immobilized anti-CD3 mAb, helper activity was strongly enhanced in the presence of these factors. This T-cell activation system may prove useful for a standardized analysis of both activation requirements and immunoregulatory capacities of human T cells.     

Inhibitory effect of 11 beta-hydroxypregna-1,4-diene-3,20-dione (delta HOP) on lymphocyte proliferation

We have compared the immunosuppressive effect of delta HOP and glucocorticoids on lymphocyte proliferation and IL-1 secretion. The new synthetic steroid only inhibited proliferation of phytohaemagglutinin (PHA)-stimulated human lymphocyte at high concentrations and the effect did not persist after washing out the steroid. In contrast, glucocorticoids produced the classical dose-response inhibition and the effect persisted when they were removed from the cultured medium. Although both steroids decreased IL-1 secretion from human monocytes stimulated with lipopolysaccharide (LPS), they exert the same effect through a different mechanism. The experiments we report suggest that the decrease of IL-1 synthesis produced by delta HOP could be caused by inhibition of LPS phagocytosis. These results support our hypothesis that delta HOP exerts its immunosuppressive and anti-inflammatory effect by a non-genomic mechanism.     

Effects of imipenem and cilastatin on human T-lymphocytes derived from acute leukemia patients with chemotherapy-induced leucopenia: studies of T-lymphocyte responses in the presence of acute myelogenous leukemia (AML) blast accessory cells

The effects of imipenem and cilastatin on human T-lymphocytes were studied in vitro. As responder T-cells were used T-lymphocyte clones derived from acute leukemia patients with chemotherapy-induced cytopenia, and the accessory cells were highly enriched acute myelogenous leukemia (AML) blasts. The effects of imipenem and cilastatin on phytohemagglutinin (PHA), anti-CD3 and anti-CD3+anti-CD28 stimulated activation were assayed, and in addition drug effects on cytokine-dependent proliferation of activated T-lymphocytes were investigated. Imipenem inhibited IL2-dependent proliferation of activated CD4(+) and CD8(+) T-cell clones, and an inhibition was also detected for IL7-, IL12-, IL15-, IL16- and IL17-dependent clonal proliferation. Imipenem caused a weak inhibition of anti-CD3- and PHA-stimulated T-cell proliferation when using 50 Gy irradiated AML blast accessory cells derived from various patients, whereas no effect was observed for anti-CD3+anti-CD28 stimulated and allostimulated activation. Imipenem decreased the release of IL4 and Interferon-gamma by T-cell clones stimulated with anti-CD3 and PHA in the presence of native (nonirradiated) AML blasts. The imipenem effects were observed at concentrations corresponding to levels reached in vivo, whereas even high concentrations of cilastatin did not alter T-cell responses. The T-lymphocyte inhibition is probably caused by a direct effect of imipenem on the T-cells.     

Isoprinosine restores in vitro T lymphocyte functions of cyclophosphamide immunosuppressed mice

The effect and the mechanism of action of isoprinosine has been investigated in several models of in vitro activation of lymphocytes. Isoprinosine added to spleen cell cultures enhanced lymphocyte proliferation induced by concanavalin A or allogeneic stimulation as well as the generation of allospecific cytotoxic T cells. The effect of isoprinosine on T lymphocyte proliferation in vitro was specially marked when mice were treated with cyclophosphamide (75-200 mg/kg) 16-24 h before the onset of cultures. No effect was observed on B cell proliferation to LPS. Addition of inosine or adenosine also enhanced proliferation of cells from both normal and cyclophosphamide treated mice. Isoprinosine and inosine and, more markedly, adenosine, augmented interleukin-2 activity in concanavalin A supernatants of spleen cells from the same animals.     

Comparative in vitro study of the immunomodulatory activity of humanized and chimeric anti-CD25 monoclonal antibodies

Humanized or chimeric monoclonal antibodies (MoAbs) directed against the interleukin-2 (IL-2) receptor alpha-chain, CD25, are promising immunosuppressive agents due to improved pharmacokinetic profiles and less toxicity. These MoAbs have been used effectively in preventing and/or treating rejection in solid organ transplantation and are currently under investigation for prevention/treatment of graft-versus-host disease (GvHD) in stem cell transplantation. We analysed the in vitro activities of the chimeric anti-CD25 MoAb basiliximab and the humanized anti-CD25 MoAb daclizumab in various test systems for alloimmune response and T cell activation in comparison to cyclosporin A (CsA) and prednisolone. Anti-CD3- and alloantigen-induced T cell proliferation were decreased significantly by the anti-CD25 MoAbs in a dose-dependent fashion. At a concentration of 10 ng/ml daclizumab and CsA synergistically decreased T cell proliferation of mixed lymphocyte cultures, whereas basiliximab showed only subadditive activity. Simultaneous addition of the anti-CD25 MoAbs and prednisolone did not result in combined activity. Addition of exogenous IL-2 completely overcame the inhibitory effect on T cell proliferation of both anti-CD25 MoAbs, but not that of CsA and prednisolone. Anti-CD25 MoAbs inhibited the generation of antigen-specific cytotoxic T lymphocytes in a limiting dilution assay, whereas they showed no effect on the cytolytic activity of established antigen-specific T cell clones. This in vitro study demonstrates strong immunosuppressive activity by both chimeric and humanized MoAbs against CD25. The combined activity with CsA justifies their early use for prevention rather than treatment of GvHD.     

Inhibition of T cell activation through down-regulation of TCR-CD3 expression mediated by an anti-CD90 Ab

We are trying to develop new Abs that can manipulate CD4 T cell responses and are usable as immunosuppressive agents. To this end, we performed functional screening, in which we examined the effect of an Ab on the proliferation of mouse CD4 T cells upon activation. The Ab, LP5, inhibited the activation of CD4 T cells stimulated with an anti-CD3 Ab or peptide antigen. The Ab alone had no stimulatory effect on CD4 T cells. Biochemical experiments demonstrated that LP5 recognized the Thy-1 (CD90) molecule. Interestingly, the treatment of CD4 T cells with LP5 in vitro induced a temporary down-regulation of CD3 expression at the cell surface. TCR molecules were also affected. Other anti-CD90 Abs not inhibitory to CD4 T cell activation failed to induce a reduction in CD3. Experiments in vitro revealed that the down-regulation caused by LP5 is due to an accelerated endocytosis of cell surface CD3. In addition, it was shown that CD3 down-regulation before or in the early stages of T cell activation is critical for the induction of hyporesponsiveness. Experiments in vivo showed that pre-treatment of CD4 T cells with LP5 inhibited the rejection of semi-allogeneic bone marrow transplants. Based on these observations, we propose that CD3 down-regulation without any stimulatory activity against T cells could be one approach to inhibiting T cell activation, and CD90 would be an appropriate target.     

ZAP-70 is required for calcium mobilization but is dispensable for mitogen-activated protein kinase (MAPK) superfamily activation induced via CD2 in human T cells

Stimulation with specific pairs of anti-CD2 antibodies can induce T cell activation and proliferation. In this study, we investigate the significance of ZAP-70 in CD2 signaling using ZAP-70-deficient T cells derived from a CD8-deficient patient and show that ZAP-70 is necessary for cellular proliferation and cytokine production in T cells stimulated via CD2. Biochemical analyses show that CD2 stimulation induces activation of mitogen-activated protein kinase (MAPK) superfamily in ZAP-70-deficient T cells, indicating that a ZAP-70-independent pathway(s) exists for MAPK superfamily activation via CD2. In contrast, intracellular Ca(2+) mobilization and activation of nuclear factor of activated T cells (NFAT) upon CD2 triggering were impaired in T cells lacking ZAP-70. Furthermore, we found that pharmacological Ca(2+) elevation combined with CD2 stimulation restored NFAT activation and subsequent cytokine production in ZAP-70-deficient T cells. These results indicate that in CD2 signaling, ZAP-70 plays an essential role in Ca(2+) mobilization and NFAT activation.     

Involvement of the CD4 molecule in a post-activation event on T cell proliferation

A monoclonal antibody (mAb) directed to a human leukocyte 55-kDa cell surface molecule with identical cellular distribution and biochemical properties to the CD4 was able to inhibit T cell proliferation induced either in a mixed lymphocyte culture or by activation with mAb anti-CD3, anti-CD2 or phytohemagglutinin. The inhibitory effect of anti-CD4 was observed in the absence of monocytes and was directly exerted on T4+ cells. This effect on cellular proliferation appears to be due to an inhibition of a postactivation event since the rise of cytoplasmic Ca2+ after activation with anti-CD3 mAb is not affected by the presence of anti-CD4 and the proliferation that occurs after an activation pulse of 3 h with ionophore and phorbol myristate acetate can be inhibited when the anti-CD4 is added after the pulse period. Kinetic studies demonstrated that the inhibition of cellular proliferation by anti-CD4 mAb was observed even if the antibody was added as late as 18-24 h after the initiation of the culture. The effect of this blocking anti-CD4 mAb on the interleukin (IL) 2/IL 2 receptor signalling pathways was also examined. The presence of anti-CD4 slightly affected the production of IL2. In fact, addition of exogenous recombinant IL2 at the initiation of the cultures did not restore the proliferative response. However, anti-CD4 had a strong inhibitory effect on the expression of IL2 receptors as analyzed by direct immunofluorescence cytometry. Taken together, these results indicate that the binding of the anti-CD4 mAb to T cells interferes with a late metabolic step being capable of abolishing the proliferative activity of fully activated cells.     

In vitro induction of regulatory T cells by anti-CD3 antibody in humans

Therapy with anti-CD3 antibody is effective in controlling models of autoimmune diseases and can reverse or prevent rejection of grafts. We studied the in vitro immunomodulatory effect of anti-CD3 treated human T cells. CD4(+) T cells were stimulated with plate-bound anti-CD3 and cultured for 12 days after which they were cultured with autologous peripheral blood mononuclear cells (PBMCs) and stimulated with soluble anti-CD3. We found that CD4(+) T cells that were stimulated with anti-CD3 (T(alphaCD3)) markedly suppressed the proliferation and cytokine production of autologous PBMCs. These regulatory T cells were not induced by incubation with isotype control (T(control)) antibody or when anti-CD3 was combined with high doses of anti-CD28 (T(alphaCD3/CD28)). T(alphaCD3) regulatory cells were anergic and produced lower levels of IFN-gamma, TNF-alpha and IL-2, and higher levels of TGF-beta than T(control) or T(alphaCD3/CD28). There were no differences in the expression of CD25 or CTLA4 on T(alphaCD3) as compared to T(control) or T(alphaCD3/CD28), and CD4(+) CD25(-) T(alphaCD3) cells were identical to CD4(+) CD25(+) T(alphaCD3) cells in their in vitro suppressive properties. Recombinant IL-2 in vitro abrogated the suppressive effect of T(alphaCD3). The suppressive effect was not related to apoptosis, was independent of HLA since T(alphaCD3) also suppressed allogeneic PBMCs, and was not related to soluble factors. Finally, no suppression was observed when non-T cells were removed from culture or when cultures were stimulated with plate-bound anti-CD3, consistent with the ability of T(alphaCD3) to downregulate CD80 on dendritic cells in co-culture experiments. Thus, we have identified human T cells with strong in vitro regulatory properties induced in vitro by anti-CD3 which appear to act in a non-HLA restricted fashion by affecting antigen presenting cells.     

Impact of 15-deoxyspergualin on effector cells in experimental autoimmune diseases of the nervous system in the Lewis rat.

The influence of the immunosuppressive antibiotic agent 15-deoxyspergualin (DSG) on macrophages and autoreactive T helper lymphocytes from Lewis rats was analysed in vitro and in vivo. DSG did not inhibit antigen- or mitogen-induced proliferation of encephalitogenic or neuritogenic T helper cell lines in vitro. However, the presence of DSG during in vitro activation of the T cells strongly suppressed or completely abrogated their capacity to induce encephalitis (EAE) or neuritis (EAN) after adoptive transfer to naive rats, although expression of activation markers or adhesion molecules on the T line blasts was not down-regulated by DSG. Like activation-induced T cell proliferation, IL-2-dependent growth of CD4+ T line cells was not affected by DSG. Preincubation of CD4+ T line cells in DSG during IL-2-driven proliferation for 48 h, however, inhibited the subsequent antigen- but not mitogen-induced activation of these T cells, although neither density of T cell receptors nor other surface molecules involved in antigen recognition were lowered on the cells exposed to DSG. Similar to its effect in vitro, in vivo administration of DSG for 10 days even at a concentration with cumulative toxicity did not suppress in vitro proliferation of spleen cells induced by mitogen or a mitogenic combination of anti-CD2 antibodies. Furthermore, spleen cell and peripheral blood lymphocyte (PBL) surface antigens, particularly MHC molecules, were not altered by long-term treatment with DSG for 30 days. While there was a slight reduction in the number of polymorphonuclear cells in both populations, the proportion of the different leucocyte subpopulations remained unchanged. In contrast to the strong functional impact of DSG on autoreactive T helper cells, the drug did not inhibit the oxidative burst of macrophages or their MHC antigen expression. This study demonstrates a clear inhibitory effect of DSG on CD4+ T lymphocytes, but not macrophages. It provides an explanation for recent observations of a strong immunosuppressive in vivo effect of DSG on transplantation rejection and experimental autoimmune diseases, despite a normal mitogen response of T cells exposed to DSG in vivo and in vitro.     

Signal transduction in lymphocyte activation through crosslinking of HLA class I molecules

The inhibitory effect of anti-HLA class I monoclonal antibodies on lymphocyte proliferation has been well documented. However, recent data suggest that anti-HLA class I monoclonal antibodies can enhance lymphocyte proliferation via both anti-CD3-induced {1,2} and anti-CD2-induced {3} activation pathways. Here we demonstrate that both inhibition and activation can be regulated by the degree of aggregation of HLA class I antigens. Crosslinking of monoclonal antibodies specific for HLA-A, HLA-B, or monomorphic determinants (using anti-IgG2 and/or anti-Ig “second step” monoclonal antibodies) increased the capacity of the anti-HLA class I monoclonal antibodies to inhibit phytohemagglutinin-induced proliferation. However, the cytosolic free calcium concentration was increased in CD4+ cells, CD8+ cells, B cells, and CD16+ cells when anti-HLA class I monoclonal antibodies were crosslinked, suggesting that an activation signal was generated by aggregation of the corresponding antigens. Indeed, inositol 1,4,5-trisphosphate could be detected in peripheral blood lymphocytes following crosslinking of anti-HLA class I monoclonal antibodies. Class I aggregation also induced proliferation of peripheral blood mononuclear cells in the presence of submitogenic doses of phorbol 12-myristate 13-acetate. Strong conditions of crosslinking (monomorphic monoclonal antibody plus both anti-IgG2 and anti-Ig ) induced CD25 expression and responsiveness to recombinant interleukin 2. Our results suggest that aggregation of HLA class I antigens primed cells to become activated in the presence of progression signals including phorbol 12-myristate 13-acetate, recombinant interleukin 2, or anti-CD5 plus anti-CD28 monoclonal antibodies.