Expression of CD26 and its Association with Dipeptidyl Peptidase IV Activity in Lymphocytes of Type 2 Diabetes Patients

Immune response and inflammation were suggested to play certain roles in the development and complications of type 2 diabetes mellitus. The main objective of this study was to investigate the CD26 expression and its relationship with adenosine deaminase (ADA), dipeptidyl peptidase IV (DPP-IV), γ-glutamyltransferase (GGT), and N-acetyl-β-glucosaminidase (NAG) activities in lymphocytes of type 2 diabetics (T2DM) patients. These parameters were assessed in 25 T2DM patients and 20 control subjects. We observed a decrease in CD26 expression and a significant increase in the ADA activity in T2DM patients when compared with control subjects. There were no differences between activities of DPP-IV, NAG, and GGT in lymphocytes of T2DM patients and control subjects. Meanwhile, a significant negative correlation was observed between CD26 expression and DPP-IV activity in lymphocytes of T2DM patients. Moreover, a positive correlation was found between DPPIV and ADA activities. The results suggest that the reduction of CD26 expression may be associated in the regulation of DPP-IV in T2DM patients.     

CD26 Expression on T Helper Populations and sCD26 Serum Levels in Patients with Rheumatoid Arthritis

We studied dipeptidyl peptidase IV (DPP-IV, CD26) expression in different T helper cells and serum soluble DPP-IV/sCD26 levels in rheumatoid arthritis (RA) patients, correlated these with disease activity score (DAS), and examined how they were affected by different therapies, conventional or biological (anti-TNF, anti-CD20 and anti-IL6R or Ig-CTLA4). The percentage of CD4⁺CD45R0⁺CD26^- cells was greatly reduced in patients (up to 50%) when compared with healthy subjects. Three other subsets of CD4 cells, including a CD26high Th1-associated population, changed variably with therapies. Data from these subsets (frequency and staining density) significantly correlated with DAS28 or DAS28 components but different in each group of patients undergoing the different therapies. Th17 and Th22 subsets were implicated in RA as independent CCR4⁺ and CCR4^- populations each, with distinct CD26 expression, and were targeted with varying efficiency by each therapy. Serum DPP-IV activity rather than sCD26 levels was lower in RA patients compared to healthy donors. DPP-IV and sCD26 serum levels were found related to specific T cell subsets but not to disease activity. We conclude that, according to their CD26 expression, different cell subsets could serve to monitor RA course, and an uncharacterized T helper CD26^- subset, not targeted by therapies, should be monitored for early diagnosis.     

CD47 signals T cell death.

Activation-induced death of T cells regulates immune responses and is considered to involve apoptosis induced by ligation of Fas and TNF receptors. The role of other receptors in signaling T cell death is less clear. In this study we demonstrate that activation of specific epitopes on the Ig variable domain of CD47 rapidly induces apoptosis of T cells. A new mAb, Ad22, to this site induces apoptosis of Jurkat cells and CD3epsilon-stimulated PBMC, as determined by morphological changes, phosphatidylserine exposure on the cell surface, uptake of propidium iodide, and true counts by flow cytometry. In contrast, apoptosis was not observed following culture with anti-CD47 mAbs 2D3 or B6H12 directed to a distant or closely adjacent region, respectively. CD47-mediated cell death was independent of CD3, CD4, CD45, or p56lck involvement as demonstrated by studies with variant Jurkat cell lines deficient in these signaling pathways. However, coligation of CD3epsilon and CD47 enhanced phosphatidylserine externalization on Jurkat cells with functional CD3. Furthermore, normal T cells required preactivation to respond with CD47-induced apoptosis. CD47-mediated cell death appeared to proceed independent of Fas or TNF receptor signaling and did not involve characteristic DNA fragmentation or requirement for IL-1beta-converting enzyme-like proteases or CPP32. Taken together, our data demonstrate that under appropriate conditions, CD47 activation results in very rapid T cell death, apparently mediated by a novel apoptotic pathway. Thus, CD47 may be critically involved in controlling the fate of activated T cells.     

The antiviral efficacy of simian immunodeficiency virus-specific CD8+ T cells is unrelated to epitope specificity and is abrogated by viral escape

CD8(+) T lymphocytes appear to play a role in controlling human immunodeficiency virus (HIV) replication, yet routine immunological assays do not measure the antiviral efficacy of these cells. Furthermore, it has been suggested that CD8+ T cells that recognize epitopes derived from proteins expressed early in the viral replication cycle can be highly efficient. We used a functional in vitro assay to assess the abilities of different epitope-specific CD8+ T-cell lines to control simian immunodeficiency virus (SIV) replication. We compared the antiviral efficacies of 26 epitope-specific CD8+ T-cell lines directed against seven SIV epitopes in Tat, Nef, Gag, Env, and Vif that were restricted by either Mamu-A*01 or Mamu-A*02. Suppression of SIV replication varied depending on the epitope specificities of the CD8+ T cells and was unrelated to whether the targeted epitope was derived from an early or late viral protein. Tat(28-35)SL8- and Gag(181-189)CM9-specific CD8+ T-cell lines were consistently superior at suppressing viral replication compared to the other five SIV-specific CD8+ T-cell lines. We also investigated the impact of viral escape on antiviral efficacy by determining if Tat(28-35)SL8- and Gag(181-189)CM9-specific CD8+ T-cell lines could suppress the replication of an escaped virus. Viral escape abrogated the abilities of Tat(28-35)SL8- and Gag(181-189)CM9-specific CD8+ T cells to control viral replication. However, gamma interferon (IFN-gamma) enzyme-linked immunospot and IFN-gamma/tumor necrosis factor alpha intracellular-cytokine-staining assays detected cross-reactive immune responses against the Gag escape variant. Understanding antiviral efficacy and epitope variability, therefore, will be important in selecting candidate epitopes for an HIV vaccine.     

Monoclonal antibodies against LFA-1 or its ligand ICAM-1 accelerate CD2 (T11.1 + T11.2)-mediated T cell proliferation

Activation of human-purified T cells can be mediated by pairwise combinations of monoclonal antibodies directed against T11.1 and T11.2 epitopes on the CD2 molecule. Monoclonal antibodies (mAbs) reactive with either the alpha and beta chains of the lymphocyte-function-associated antigen-1 (LFA-1) molecule or one of its ligands, intercellular adhesion molecule-1 (ICAM-1), were found to accelerate anti-CD2-induced proliferation. This effect was seen on thymocytes and resting or preactivated T cells (phytohemagglutinin blasts and alloproliferative T cell clones) and could be observed, following the introduction of anti-LFA-1 or -ICAM-1 mAbs, up to 50 hr after the CD2 stimulatory signal. This effect was equally abrogated by 55 kDa anti-interleukin-2 (IL-2) receptor mAb, but neither the expression of IL-2 receptor nor the production of IL-2 was modified. The effects of anti-LFA-1 or anti-ICAM-1 on T cell activation through the CD2 pathway were therefore opposite to those observed in the CD3 pathway, where both mAbs strongly delayed T cell proliferation.     

Human T cell activation by OKT3 is inhibited by a monoclonal antibody to CD44.

The CD44 molecule, also known as Hermes lymphocyte homing receptor, human Pgp-1, and extracellular matrix receptor III, has been shown to play a role in T cell adhesion and activation. Specifically, anti-CD44 mAb block binding of lymphocytes to high endothelial venules, inhibit T cell-E rosetting, and augment T cell proliferation induced by the CD2 or CD3-TCR pathways. We have characterized an anti-CD44 mAb (212.3) which immunoprecipitates a 90-kDa protein and is specific for CD44 as shown by peptide mapping and antibody competition studies. Interestingly, our studies with 212.3 demonstrate that this CD44-specific mAb completely inhibits T cell proliferation stimulated by the anti-CD3 mAb, OKT3. Inhibition is not a result of reduced cell viability, but is associated with 1) inhibition of IL-2 production, 2) inhibition of IL-2R expression, and 3) inhibition of OKT3-mediated increases in intracellular Ca2+ levels. In addition, 212.3 does not inhibit proliferation by the T cell mitogens PHA or PWM nor does it inhibit proliferation in a mixed lymphocyte reaction. Similar to other anti-CD44 mAb, 212.3 also augments T cell proliferation induced by mAb directed against the T11(2) and T11(3) epitopes of CD2. Thus, these studies describe a novel CD44-specific mAb (212.3) that inhibits T cell activation by OKT3 by blocking early signal transduction. Furthermore, these studies suggest that "receptor cross-talk" between the CD3-TCR complex and CD44 may regulate T cell activation.     

Cloning and functional expression of the T cell activation antigen CD26.

A cDNA encoding the T cell activation Ag CD26 was isolated from human PHA-activated T cells by using an expression cloning method. The nucleotide sequence obtained predicts a protein of 766 amino acids of type II membrane topology, with six amino acids in the cytoplasmic region. The predicted amino acid sequence of the Ag was 85% homologous to that of the dipeptidyl peptidase IV enzyme isolated from rat liver. Derivatives of the human leukemic T cell line Jurkat transfected with a CD26 expression plasmid were established. Characterization of the CD26 Ag expressed by the transfected Jurkat cells revealed that the Ag could be immunoprecipitated as a 110-kDa molecule similar to that found on peripheral blood T cells and that the Ag had dipeptidyl peptidase IV activity. Functional analysis of these Jurkat transfectants showed that cross-linking of the CD26 and CD3 Ag with their respective antibodies resulted in enhanced intracellular calcium mobilization and IL-2 production. These results provide direct evidence that the CD26 Ag plays a role in T cell activation.     

Expression and characterization of a novel CD6 ligand in cells derived from joint and epithelial tissues

CD6 is a T cell surface glycoprotein that plays an important role in interactions of thymocytes with thymic epithelial cells and in mature T cell interactions with selected nonprofessional tissue APCs. We describe a novel CD6 ligand (CD6L) 3A11 Ag that is distinct from the known CD6L (CD166). The 3A11 protein is expressed on cells derived from human thymus, skin, synovium, and cartilage, and its expression is enhanced by IFN-gamma. mAbs directed against the 3A11 Ag and CD166 exhibit distinct patterns of binding to a panel of cell lines. Confocal microscopy shows that both CD166 and the 3A11 Ag are expressed at the cell surface, and that these proteins colocalize. The 3A11 Ag has a molecular mass of 130 kDa and is immunoprecipitated using either mAb 3A11 or soluble CD6-Ig fusion protein. mAbs directed against individual CD6L were less potent than was soluble CD6-Ig fusion protein in reducing adhesion of T cells to adherent 3A11-positive epithelial cells in vitro, suggesting that these Abs recognize epitopes on the 3A11 Ag and CD166 that are distinct from CD6 binding sites. Finally, transfection of epithelial cells with CD166-specific small interfering RNAs significantly decreased CD166 expression without alteration in 3A11 Ag levels, and thus confirmed that these two CD6L are distinct. Taken together, our data identifies a novel 130-kDa CD6L that may mediate interactions of synovial and epithelial cells with T lymphocytes.     

The HIV-1 gp120 inhibits the binding of adenosine deaminase to CD26 by a mechanism modulated by CD4 and CXCR4 expression

HIV-1 external envelope glycoprotein gp120 inhibits adenosine deaminase (ADA) binding to its cell surface receptor in lymphocytes, CD26, by a mechanism that does not require the gp120-CD4 interaction. To further characterize this mechanism, we studied ADA binding to murine clones stably expressing human CD26 and/or human CD4, and transiently expressing human CXCR4. In this heterologous model, we show that both recombinant gp120 and viral particles from the X4 HIV-1 isolate IIIB inhibited the binding of ADA to wild-type or catalytically inactive forms of CD26. In cells lacking human CXCR4 expression, this gp120-mediated inhibition of ADA binding to human CD26 was completely dependent on the expression of human CD4. In contrast, when cells were transfected with human CXCR4 the inhibitory effect of gp120 was significantly enhanced and was not blocked by anti-CD4 antibodies. These data suggest that the interaction of gp120 with CD4 or CXCR4 is required for efficient inhibition of ADA binding to CD26, although in the presence of CXCR4 the interaction of gp120 with CD4 may be dispensable.     

Role of the CD45 (T-200) molecule in anti-CD3-triggered T cell-mediated cytotoxicity

NK-depleted human peripheral blood lymphocytes can be modulated with anti-CD3 to kill certain targets during 3-hr cytotoxicity assays. When triggered by anti-CD3 antibody, these effector T cells killed only NK-sensitive targets, such as K562 and HEL 92.1.7, and NK-resistant targets, such as Daudi, whose killing is inhibited by anti-CD45 (T-200) monoclonal antibodies, such as 13.3. NK-sensitive targets, MOLT-4, U266/AF10, Jurkat, and CCFR-CEM, and 10 NK-resistant cell lines, including Raji, IM-9, U698, U937, and GM-1056, whose killing is not inhibited by anti-CD45 monoclonal antibodies, were not killed by alpha-CD3-T effectors, suggesting that the CD45 molecule may be involved in the killing process. Anti-CD3-triggered T cell killing of target cells was inhibited greater than 95% by the monoclonal antibody 13.3. This inhibition of cytotoxicity by 13.3 was not due to competition of this IgG1 antibody for Fc receptor binding site on the target cell, since the IgG1 monoclonal antibody anti-beta 2-microglobulin did not block cytotoxicity. Single cell assays and calcium pulse assays showed that CD45 is involved in a postbinding, pre-calcium-dependent stage, similar to that shown for NK cytotoxicity. There was a relative shift of importance of different epitopes of CD45 in anti-CD3-T cytotoxicity compared to NK cytotoxicity. Anti-CD45 antibodies which bind to the C terminus end of the molecule played a more important role in anti-CD3-T cytotoxicity than NK cytotoxicity. Thus, a subset of T cells exists that exhibits anti-CD3-triggered non-MHC-restricted killing of certain NK-sensitive and NK-resistant targets in association with a CD45 molecule which is functionally different from the NK CD45 molecule.     

Cholera toxin inhibits the increase in cytoplasmic free calcium induced via the CD2 pathway of human T-lymphocyte activation

We investigated the action of cholera toxin on the intracellular ionized calcium [Ca2+]i increase induced by anti-CD2 and anti-CD3 monoclonal antibodies in the leukemic human T-cell line Jurkat. Cholera toxin inhibits in a dose-dependent manner these two pathways of human T-lymphocyte activation but with different half maximal inhibition doses (75 ng/ml for CD3, 30 ng/ml for CD2). This effect cannot be accounted for only by the increase in cAMP induced by cholera toxin because forskolin, which raises cellular cyclic adenosine monophosphate (cAMP) to the same levels, induced only a small inhibition of the [Ca2+]i increase in similar conditions. Cholera toxin induced a decrease in the surface expression of the CD3 molecule, suggesting a down-regulation of the CD3 molecules. On the other hand, the expression of CD2 remained unchanged. Cell surface disappearance of the CD3 molecule cannot account for all the inhibitory effects of cholera toxin because CD2 molecule expression was not affected (no modifications in the half maximal binding of anti-CD2 monoclonal antibodies). All together, these results suggest that cholera toxin acts on substrates, possibly G proteins, that could regulate the [Ca2+]i increase induced by anti-CD2 and anti-CD3 mAbs in Jurkat cells. In addition, the present study demonstrated that the rise in cellular cAMP partially inhibits the [Ca2+]i increase induced by anti-CD2 and anti-CD3 mAbs.     

一种新型的重组单链三特异抗体的构建与表达

双特异抗体由于其缺乏共刺激信号 ,激活的T细胞往往会导致凋亡。为了更有效地杀伤肿瘤细胞 ,构建了抗卵巢癌单链×抗CD3单链×抗CD2 8单域重组三特异抗体的表达载体。利用大肠杆菌中的分子伴侣FkpA与三特异抗体共表达 ,提高了三特异抗体的在BL2 1Star菌中的可溶性原核表达。ELISA结果显示 ,可溶性的重组单链三特异抗体 (sTRI)与SK OV 3细胞的膜抗原 ,Jurkat细胞上的CD3分子以及重组CD2 8抗原均有较强的结合活性。桥连试验证明该抗体能同时与SK OV 3细胞和Jurkat细胞结合。体外杀伤试验表明该抗体能激活外周血T细胞来杀伤肿瘤细胞。形态学观察也进一步说明该抗体具有良好的结合活性以及体外杀伤活性。这种新型的重组单链三特异抗体为基于T细胞的癌症免疫治疗建立了一个新的技术平台。     

Involvement of TNF-related apoptosis-inducing ligand in human CD4+ T cell-mediated cytotoxicity

TNF-related apoptosis-inducing ligand (TRAIL) has been identified as a member of the TNF family that induces apoptosis in a variety of tumor cells, but its physiological functions are largely unknown. In the present study, we examined the expression and function of TRAIL in human CD4+ T cell clones by utilizing newly established anti-human TRAIL mAbs. Human CD4+ T cell clones, HK12 and 4HM1, exhibited perforin-independent and Fas ligand (FasL)-independent cytotoxicity against certain target cells, including T lymphoma (Jurkat) and keratinocyte (HaCaT) cell lines, which are susceptible to TRAIL-mediated cytotoxicity. In contrast to FasL, the expression of which was inducible upon anti-CD3 stimulation, TRAIL was constitutively expressed on HK12 and 4HM1 cells, and no further increase was observed after anti-CD3 stimulation. Spontaneous cytotoxic activities of resting HK12 and 4HM1 cells against Jurkat and HaCaT cells were blocked by anti-TRAIL mAb but not by anti-FasL mAb, and bystander cytotoxic activities of anti-CD3-stimulated HK12 and 4HM1 cells were abolished by the combination of anti-TRAIL and anti-FasL mAbs. These results indicate a differential regulation of TRAIL and FasL expression on human CD4+ T cell clones and that TRAIL constitutes an additional pathway of T cell-mediated cytotoxicity.     

CD200 is a ligand for all members of the CD200R family of immunoregulatory molecules

CD200Fc, a chimeric molecule including the extracellular domain of CD200 and a murine IgG2a Fc region, regulates immune responses following engagement of a cell surface receptor, CD200R, expressed on cells of the myeloid and T cell lineage. A recent report focused attention on a family of CD200Rs, but concluded that only one member used CD200 as its ligand. We have also cloned and sequenced a family of CD200Rs, but identify an amino terminus to two of the three isoforms not recognized by previous researchers. We show by FACS, using FITC-labeled CD200Fc, that COS7 cells transfected with all CD200R isoforms bind CD200 as ligand, although the functional consequences of this binding likely differs between the different isoforms. mAbs directed against the CD200 R1/R4 isoforms altered IL-2/IL-4 cytokine production and suppressed CTL responses in a fashion comparable to CD200Fc, with a significantly lesser effect seen following addition of anti-CD200 R2/R3.     

Properties of anti-gp41 core structure antibodies, which compete with sera of HIV-1-infected patients

To determine the correlation between the immunoreaction against the core structure of human immunodeficiency virus type (HIV-1) transmembrane protein gp41 epitopes and the disease progression, it is essential to evaluate the anti-core structure antibody epitopes and the humoral immunity against the epitopes. For this purpose we evaluated monoclonal antibodies (mAbs) against the gp41 core structure such as mAbs 50.69, 98.6 and T26, by Western blotting (WB) and flow cytometry. WB showed mAbs 50.69 and 98.6 bound to both monomeric and oligomeric gp41, and mAb T26 exclusively bound to oligomeric gp41. We evaluated the sera from Pneumocystis pneumonia patients (PCP; n=7) and long-term survivors (LTS; n=7). Competition assay with sera and mAbs for binding to H9 cells infected with HIV-1 IIIB virus was done using flow cytometry. The results revealed that PCP sera as well as LTS sera inhibited the binding of all the three mAbs, and the PCP sera inhibited mAb T26 binding more efficiently than LTS. Therefore, PCP patients retain competing immunity to antibodies against not only the shared epitopes of the core structure (binding sites of mAbs 50.69 and 98.6) but also against oligomeric gp41 specific epitope (binding site of mAb T26).     

Comprehensive analysis of human immunodeficiency virus type 1 (HIV-1)-specific gamma interferon-secreting CD8+ T cells in primary HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1)-specific CD8(+) T cells provide an important defense in controlling HIV-1 replication, particularly following acquisition of infection. To delineate the breadth and potency of these responses in patients upon initial presentation and before treatment, we determined the fine specificities and frequencies of gamma interferon (IFN-gamma)-secreting CD8(+) T cells recognizing all HIV-1 proteins in patients with primary infection. In these subjects, the earliest detected responses were directed predominantly against Nef, Tat, Vpr, and Env. Tat- and Vpr-specific CD8(+) T cells accounted for the greatest frequencies of mean IFN-gamma spot-forming cells (SFC). Nef-specific responses (10 of 21) were more commonly detected. A mean of 2.3 epitopes were recognized with various avidities per subject, and the number increased with the duration of infection (R = 0.47, P = 0.031). The mean frequency of CD8(+) T cells (985 SFC/10(6) peripheral blood mononuclear cells) correlated with the number of epitopes recognized (R = 0.84, P < 0.0001) and the number of HLA-restricting alleles (R = 0.79, P < 0.0001). Neither the total SFC frequencies nor the number of epitopes recognized correlated with the concurrent plasma viral load. Seventeen novel epitopes were identified, four of which were restricted to HLA alleles (A23 and B72) that are common among African descendents. Thus, primary HIV-1 infection induces strong CD8(+)-T-cell immunity whose specificities broaden over time, but their frequencies and breadth do not correlate with HIV-1 containment when examined concurrently. Many novel epitopes, particularly directed to Nef, Tat, and Env, and frequently with unique HLA restrictions, merit further consideration in vaccine design.     

Triggering of the proteinase dipeptidyl peptidase IV (CD26) amplifies human T lymphocyte proliferation.

CD26 (Ta1, dipeptidyl peptidase IV) is a Mr 105,000 protein expressed at high levels on activated T lymphocytes and is a potential marker of memory T cells. Reciprocal immunodepletion and solid phase double determinant binding studies showed that mAb AC7 and the CD26-specific mAb anti-Ta1 reacted with spatially distinct sites on the same molecule. The proteinase dipeptidyl peptidase IV (DPP IV) was immunoprecipitated with mAb AC7 and its enzymatic activity directly assayed using an enzyme overlay membrane system. High levels of DPP IV activity were detected on the T cell tumor line CCRF-HSB-2 and on PBMC stimulated by a variety of methods. By itself, soluble mAb AC7 was not mitogenic for T cells but enhanced T cell proliferation that resulted from treatment with phorbol myristic acetate (PMA) in the presence of accessory cells. T cell proliferation was also induced by co-immobilized mAb AC7 and mAb OKT3 (anti-CD3). Cultures of T cells growing in the presence of IL-2 responded with accelerated growth when exposed to a combination of immobilized mAb AC7 and soluble mAb OKT3, a result not seen with freshly isolated T cells.     

Role of CD2 in regulation of CD3- LGL function.

CD2 is a differentiation marker present on T cells and NK cells. Cytotoxic T lymphocytes (CTL) can be activated by antibodies directed against the CD3/T-cell receptor complex and CD2 structures; however, the role of CD2 in regulation of CD3- large granular lymphocyte (LGL) functions has only recently been studied. Anti-CD2 monoclonal antibodies (mAbs) may be either augmenting or inhibitory and T-cell activation via the CD2 molecule occurs only when mAb binds defined combinations of the CD2 epitopes. Since LGL can be activated by a single stimulus (e.g., IL-2) to proliferate, produce IFN gamma, and increase their cytolytic potential, these functions were chosen to examine the effects of the anti-CD2 mAb and its combinations. Anti-CD2 mAb (D66, GT2, and X11-1) were incubated with LGL for various times in the absence or presence of IL2 and IFN gamma production was monitored. Single anti-CD2 mAb treatment demonstrated minimal augmentation of IFN gamma production. However, combinations of anti-CD2 (9.6) and the other anti-CD2 mAb resulted in a significant, synergistic enhancement of the IFN gamma production. Anti-CD2 mAb treatment appeared to inhibit production generated by optimal doses of IL-2 (1,000 U/ml). The effect of anti-CD2 mAb on IFN gamma production parallel their effects on LGL NK and LAK activity. These data suggested that mAb against the CD2 molecule were important in regulating LGL functions in the absence of a functional CD3 receptor in LGL.(ABSTRACT TRUNCATED AT 250 WORDS)     

T cell-specific adapter protein inhibits T cell activation by modulating Lck activity

We previously reported the isolation of a cDNA encoding a T cell-specific adapter protein (TSAd). Its amino acid sequence contains an SH2 domain, tyrosines in protein binding motifs, and proline-rich regions. In this report we show that expression of TSAd is induced in normal peripheral blood T cells stimulated with anti-CD3 mAbs or anti-CD3 plus anti-CD28 mAbs. Overexpression of TSAd in Jurkat T cells interfered with TCR-mediated signaling by down-modulating anti-CD3/PMA-induced IL-2 promoter activity and anti-CD3 induced Ca2+ mobilization. The TCR-induced tyrosine phosphorylation of phospholipase C-gamma1, SH2-domain-containing leukocyte-specific phosphoprotein of 76kDa, and linker for activation of T cells was also reduced. Furthermore, TSAd inhibited Zap-70 recruitment to the CD3zeta-chains in a dose-dependent manner. Consistent with this, Lck kinase activity was reduced 3- to 4-fold in COS-7 cells transfected with both TSAd and Lck, indicating a regulatory effect of TSAd on Lck. In conclusion, our data strongly suggest an inhibitory role for TSAd in proximal T cell activation.     

Efficiency of T cell triggering by anti-CD3 monoclonal antibodies (mAb) with potential usefulness in bispecific mAb generation

 T cell triggering can be achieved by monoclonal antibodies (mAbs) specific for the CD3/TcR complex. In the presence of appropriate costimulation and/or progression factors, such triggering permits the generation of effector cells for immunotherapy protocols involving the redirection of T cell lysis against tumor cells by mAbs bispecific for anti-CD3/anti-tumor cells (bs-mAbs). Focusing our analysis on the clinically relevant bs-mAb OC/TR, we found that bs-mAbs generated with the same anti tumor specificity, but two other anti-CD3 mAbs, TR66 and OKT3, have the same and a significantly lower lytic potential, respectively, compared with that of OC/TR. To evaluate the relevance of the anti-CD3 component, we examined several anti-CD3 mAbs with respect to binding parameters and the ability to trigger T lymphocytes. Competitive binding assays suggested that all anti-CD3 mAbs recognized the same or overlapping epitopes, although mAbs BMA030 and OC/TR bound with lower avidity than did αCD3 (the bivalent anti-CD3 mAb produced by the hybrid hybridoma OC/TR), TR66 and OKT3, as determined by measurement of the affinity constants. In all lymphocyte populations examined, which included resting peripheral blood mononuclear cells (PBMC), activated PBMC and T cell clones, OKT3, BMA033 and OC/TR failed to mobilize Ca²⁺ without cross-linking, whereas αCD3, in both murine and murine-human chimeric versions, TR66 and BMA030, did not require cross-linking. The ability to induce CD3 modulation was associated in part with the induction of Ca²⁺ fluxes. Despite the differences in the behavior of these mAbs in triggering the events that precede proliferation, all of them ultimately led to expression of the IL-2 receptor and to proliferation in T cells in the presence of accessory cells. Our data suggest that anti-CD3 mAbs that bind more rapidly (strong Ca²⁺ mobilizers) and more tightly under physiological conditions are good candidates for retargeting T cells in the bs-mAb clinical application. Received: 2 January 1997 / Accepted: 6 February 1997