Presence Of anti-CD3 Monoclonal Antibody Research Articles

Human umbilical vein endothelial cells promote the inhibitory activation of CD4+CD25+Foxp3+ regulatory T cells via PD-L1

BackgroundAtherosclerosis (AS) is a chronic inflammation characterized by massive infiltration of inflammatory cells in arterial wall plaques. Programmed death ligand-1 (PD-L1), a co-stimulatory molecule, plays a vital role in regulating immune responses. We investigated the role and mechanisms of PD-L1 expressed on oxidized low-density lipoprotein (ox-LDL)-impaired human umbilical vein endothelial cells (HUVECs) in promoting activation and cytokine production of CD4+CD25+ forkhead box P3 (FoxP3) regulatory T cells (Tregs). Methods and resultsTregs were incubated alone, with HUVECs or HUVECs pre-stimulated with ox-LDL in the presence of anti-CD3 monoclonal antibodies (mAbs) for 48 h. HUVECs were shown to upregulate the immune phenotypic markers of Tregs, such as glucocorticoid-induced TNF receptor (GITR), cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death-1 protein (PD-1). Moreover, HUVECs modulated cytokine production of Tregs (e.g., interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1)). HUVECs treated with anti-PD-L1 mAbs were unable to regulate the surface expression and cytokine production of Tregs. The Transwell culture system suggested that interaction between HUVECs and Tregs via PD-L1 requires cell-to-cell contact. ConclusionExpression of the negative co-stimulatory molecule PD-L1 on HUVECs may upregulate the inhibitory activation and cytokine production of CD4+CD25+Foxp3+ regulatory T cells in AS.

Ascorbic acid promotes proliferation of natural killer cell populations in culture systems applicable for natural killer cell therapy

Background aimsNatural killer (NK) cell–based immunotherapy is a promising treatment for a variety of malignancies. However, generating sufficient cell numbers for therapy remains a challenge. To achieve this, optimization of protocols is required. MethodsMature NK cells were expanded from peripheral blood mononuclear cells PBMCs in the presence of anti-CD3 monoclonal antibody and interleukin-2. Additionally, NK-cell progenitors were generated from CD34+ hematopoietic stem cells or different T/NK-cell progenitor populations. Generated NK cells were extensively phenotyped, and functionality was determined by means of cytotoxicity assay. ResultsAddition of ascorbic acid (AA) resulted in more proliferation of NK cells without influencing NK-cell functionality. In more detail, PBMC-derived NK cells expanded 2362-fold (median, range: 90–31,351) in the presence of AA and were capable of killing tumor cells under normoxia and hypoxia. Moreover, hematopoietic stem cell–derived progenitors appeared to mature faster in the presence of AA, which was also observed in the NK-cell differentiation from early T/NK-cell progenitors. ConclusionsMature NK cells proliferate faster in the presence of phospho-L-AA, resulting in higher cell numbers with accurate functional capacity, which is required for adoptive immunotherapy.

Regulatory T Cells Protect Fine Particulate Matter-Induced Inflammatory Responses in Human Umbilical Vein Endothelial Cells

Objective. To investigate the role of CD4+CD25+ T cells (Tregs) in protecting fine particulate matter (PM-) induced inflammatory responses, and its potential mechanisms. Methods. Human umbilical vein endothelial cells (HUVECs) were treated with graded concentrations (2, 5, 10, 20, and 40 µg/cm2) of suspension of fine particles for 24h. For coculture experiment, HUVECs were incubated alone, with CD4+CD25− T cells (Teff), or with Tregs in the presence of anti-CD3 monoclonal antibodies for 48 hours, and then were stimulated with or without suspension of fine particles for 24 hours. The expression of adhesion molecules and inflammatory cytokines was examined. Results. Adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), and inflammatory cytokines, such as interleukin (IL-) 6 and IL-8, were increased in a concentration-dependent manner. Moreover, the adhesion of human acute monocytic leukemia cells (THP-1) to endothelial cells was increased and NF-κB activity was upregulated in HUVECs after treatment with fine particles. However, after Tregs treatment, fine particles-induced inflammatory responses and NF-κB activation were significantly alleviated. Transwell experiments showed that Treg-mediated suppression of HUVECs inflammatory responses impaired by fine particles required cell contact and soluble factors. Conclusions. Tregs could attenuate fine particles-induced inflammatory responses and NF-κB activation in HUVECs.

Generation and expansion of regulatory human CD4 + T-cell clones specific for pancreatic islet autoantigens

Autoantigen-specific regulatory T cells (Treg) are a potential cell therapy for human autoimmune disease, provided they could be generated in adequate numbers and with stable function. To this end, we determined the feasibility of cloning and expanding human CD4 + Treg specific for the type 1 diabetes autoantigens, GAD65 and proinsulin. Blood CD4 + cells stimulated to divide in response to GAD65 (in three healthy individuals) or proinsulin (in one type 1 diabetic) were flow sorted into single cells and cultured on feeder cells in the presence of anti-CD3 monoclonal antibody, IL-2 and IL-4. Clones were expanded over 4–6 weeks and screened for autoantigen-dependent suppression of tetanus toxoid-specific T-cell proliferation. Suppression by Treg clones was then confirmed against autoantigen-specific non-Treg clones. Of a total of 447 clones generated, 98 (21.9%) had autoantigen-dependent suppressor function. Treg clones were anergic but proliferated to autoantigen after addition of IL-2 or in co-culture with stimulated bulk T cells, without loss of suppressor function. Treg clones were stored over liquid N 2, thawed and further expanded over 12 days, whereupon they exhibited decreased suppressor function. Expansion of Treg clones overall was in the order 10 7–10 8-fold. Treg clones were not distinguished by markers of conventional CD4 +CD25 + Treg and suppressed independently of cell–cell contact but not via known soluble suppressor factors. This study demonstrates that autoantigen-specific CD4 + Treg clones with potential application as a cell therapy for autoimmune disease can be generated and expanded from human blood.

Expression of fusion IL2-B7.1(IgV+C) and effects on T lymphocytes

The search for an effective immunotherapeutic treatment for tumors is an important area of cancer research. To prepare a more effective form of the bifunctional fusion protein IL2-B7.1(IgV+C) and analyze its effect on the stimulation of T lymphocyte proliferation, we used DNAStar 5.03 software to predict the structural diversity and biochemical character of IL2-B7.1(IgV+C). We then prepared fusion protein IL2-B7.1(IgV+C) by establishing its prokaryotic expression system, and tested its effect on the stimulation of T lymphocytes in vitro. The results indicated that IL2-B7.1(IgV+C) correctly formed a secondary structure in which both IL2 and B7.1(IgV+C) maintained their original hydrophilicity and epitopes. Western blot analysis revealed that IL2-B7.1(IgV+C) was efficiently expressed. Our analysis of CTLL-2 and T-cell proliferation showed that recombinant human (rh) IL2-B7.1(IgV+C) exerted the combined stimulating effects of both rhIL2 and rh B7.1(IgV+C) on cell proliferation, and that these effects could be blocked by adding either anti-IL2 or anti-B7.1 monoclonal antibodies. A >2-fold increase in [3H]TdR incorporation compared with that of cells treated with recombinant protein IL2, or B7.1(IgV+C) alone, revealed that rhIL2-B7.1(IgV+C) had dose-dependent synergetic effects on T-cell activation in the presence of anti-CD3 monoclonal antibody. We concluded that the augmented potency of rhIL2-B7.1(IgV+C) resulted in a stronger stimulation of T-cell proliferation than either rhB7.1(IgV+C) or rhIL2 alone.

Effects of dexamethasone on interleukin-10R1 expression on human CD4+ T cells.

In his interesting letter, Dr. Visser stresses the concept that “under GC pressure, functional expression of the IL-10 receptor must remain in order to allow synergy between GC and IL-10.” This statement poses some important questions: (1) Is interleukin (IL)-10R resistant to glucocorticoid (GC) pressure? (2) If not, which degree of inhibition is compatible with the synergism? (3) Which roles do IL-10R1 and IL-10R2 play in the synergizing activity of IL-10? (4) Does the duration of GC pressure condition IL-10R expression in vivo? Our unpublished data can answer, at least in part, the first two questions. We have studied IL-10R1 expression on CD4+ T cells cultured for 24 hr with or without dexamethasone (1–100 nM), and on CD4+ T cells cultured for 12 to 72 hr in the presence of anti-CD3 monoclonal antibody, with or without dexamethasone (1–100 nM). Dexamethasone did not decrease IL-10R1 expression on unstimulated CD4+ T cells. Anti-CD3 stimulation enhanced IL-10R1 expression. Dexamethasone partly decreased it, in a dose-dependent manner, reaching the maximum effect at a concentration of 100 nM with a mean percentage of inhibition of approximately 30% at all time points studied. Therefore, IL-10R1 expression on CD4+ T cells is relatively resistant to GC, and the degree of GC resistance depends on the functional state of the cells. Moreover, the synergism seems to be compatible with a certain degree of inhibition of IL-10R1 expression. Answering the other questions will allow a more rational approach to the development of therapeutic protocols based on the use of GC in combination with IL-10. Mauro Brunetti Francesca B. Aiello

Activation-Induced Expression of Cell Surface CD28 on Mouse T Lymphocytes is Inhibited by Cyclosporine A

T-cell activation requires both T-cell receptor signaling and a costimulatory signal provided by CD28 which enhances and prolongs interleukin-2 (IL-2) production. To determine the effect of cyclosporine A (CsA) on constitutive and activation-induced CD28 expression, mouse T cells were exposed to CsA (0.1 μm) in the absence or presence of anti-CD3 monoclonal antibody (mAb). CD28 expression was then determined by flow cytometry. CsA treatment prevented activation-induced CD28 expression but did not affect constitutive CD28 expression. Inhibition of inducible CD28 expression by CsA was not rapidly reversible, requiring 48 h of restimulation in the absence of CsA for CD28 expression to return to control levels. T cells activated in the presence of combined anti-IL-2 and anti-CD25 mAb (both 10 μg/mL) also exhibited reduced CD28 expression, suggesting that activation-induced CD28 expression is, at least in part, an IL-2-dependent process. However, the inhibitory effect of CsA on activation-induced CD28 expression was maintained in the presence of exogenous IL-2 (250 U/mL). We conclude that CsA, by inhibiting activation-induced expression of costimulatory CD28 molecules by T lymphocytes, may interfere with the ability of CD28 to provide an optimal costimulatory signal for sustained IL-2 production following T-cell activation.

In vivo administration of IL-18 can induce IgE production through Th2 cytokine induction and up-regulation of CD40 ligand (CD154) expression on CD4+ T cells.

IL-18 is considered to be a strong cofactor for CD4+ T helper 1 (Th1) cell induction. We have recently reported that IL-18 can induce IL-13 production in both NK cells and T cells in synergy with IL-2 but not IL-12, suggesting IL-18 can induce Th1 and Th2 cytokines when accompanied by the appropriate first signals for T cells. We have now found that IL-18 can act as a cofactor to induce IL-4, IL-10 and IL-13 as well as IFN-gamma production in T cells in the presence of anti-CD3 monoclonal antibodies (mAb). IL-18 can rapidly induce CD40 ligand (CD154) mRNA and surface expression on CD4+ but not CD8+ T cells. The administration of IL-18 alone in vivo significantly increased serum IgE levels in C57BL/6 (B6) and B6 IL-4 knockout mice. Furthermore, the administration of IL-18 plus IL-2 induced approximately 70-fold and 10-fold higher serum levels of IgE and IgG1 than seen in control B6 mice, respectively. IgE and IgG1 induction in B6 mice by administration of IL-18 plus IL-2 was eliminated by the pretreatment of mice with anti-CD4 or anti-CD154, but not anti-CD8 or anti-NK1.1 mAb. These results suggest that IL-18 can induce Th2 cytokines and CD154 expression, and can contribute to CD4+ T cell-dependent, IL-4-independent IgE production.

Identification of a Novel, Dendritic Cell-associated Molecule, Dectin-1, by Subtractive cDNA Cloning

Dendritic cells (DC) are special subsets of antigen presenting cells characterized by their potent capacity to activate immunologically naive T cells. By subtracting the mRNAs expressed by the mouse epidermus-derived DC line XS52 with the mRNAs expressed by the J774 macrophage line, we identified five novel genes that were expressed selectively by this DC line. One of these genes encoded a type II membrane-integrated polypeptide of 244 amino acids containing a putative carbohydrate recognition domain motif at the COOH-terminal end. This molecule, termed "dectin-1," was expressed abundantly at both mRNA and protein levels by the XS52 DC line, but not by non-DC lines (including the J774 macrophage line). Dectin-1 mRNA was detected predominantly in spleen and thymus (by Northern blotting) and in skin-resident DC, i.e. Langerhans cells (by reverse transcription-polymerase chain reaction). Affinity-purified antibody against dectin-1 identified a 43-kDa glycoprotein in membrane fractions isolated from the XS52 DC line and from the dectin-1 cDNA-transfected COS-1 cells. His-tagged recombinant proteins containing the extracellular domains of dectin-1 showed marked and specific binding to the surface of T cells and promoted their proliferation in the presence of anti-CD3 monoclonal antibody at suboptimal concentrations. These in vitro results suggest that dectin-1 on DC may bind to as yet undefined ligand(s) on T cells, thereby delivering T cell co-stimulatory signals. Not only do these results document the efficacy of subtractive cDNA cloning for the identification of unique genes expressed by DC, they also provide a framework for studying the physiological function of dectin-1.

Effect of Exopolysaccharide V2-7, Isolated from Halomonas Eurihalina, on the Proliferation In Vitro of Human Peripheral Blood Lymphocytes

The immunomodulatory activity of the exopolysaccharide V2-7, a sulfated polymer excreted by the moderately halophilic bacteria Halomonas eurihalina, was studied in vitro. [3H]thymidine incorporation and flow-cytometry measurements showed that this exopolysaccharide enhanced the unspecific proliferation of human lymphocytes in response to the presence of anti-CD3 monoclonal antibody. It was effective at concentrations of less than lμg/ml, maximum activity being achieved at 0.2μ-g/ml,

Identification and characterization of an alternative cytotoxic T lymphocyte-associated protein 4 binding molecule on B cells.

To determine whether alternative cytotoxic T lymphocyte-associated protein 4 (CTLA4) binding proteins exist on B cells, we constructed (i) mCTLA4hIgG consisting of the extracellular region of a mouse CTLA4 molecule and the Fc portion of a human IgG1 molecule and (ii) PYAAhIgG, a mutant mCTLA4hIgG, having two amino acid substitutions on the conserved MYPPPY motif in the complementarity-determining region 3-like region and lacking detectable binding to both B7-1 and B7-2 molecules. Using these fusion proteins (mCTLA4hIgG and PYAAhIgG), we demonstrated that a mouse immature B-cell line, WEHI231 cells, expressed alternative CTLA4 binding molecules (ACBMs) that were distinct from both B7-1 and B7-2. ACBMs were 130-kDa disulfide-linked proteins. More importantly, ACBMs were able to provide costimulatory signal for T-cell proliferation in the presence of anti-CD3 monoclonal antibodies. In addition, we demonstrated that more than 20% of B220+ cells obtained from normal mouse spleen expressed ACBMs.

Natural cytotoxic T cells responsible for anti-CD3-induced cytotoxicity in mice.

T cells obtained from normal mouse spleen cells showed significant cytotoxic activity against Fc receptor positive tumor cells in the presence of anti-CD3 monoclonal antibody (mAb). This activity was designated as natural cytotoxic T cell (NCT) activity and compared with natural killer (NK) activity. Considerable levels of NCT activity were detected in mouse strains with both high and low NK activity. NCT cells were distributed in both lower and higher density fractions of Percoll discontinuous density gradients, while NK cells were enriched in the lower density fraction of Percoll gradients. Moreover, NCT activity was resistant to in vivo anti-asialo GM1 treatment, in contrast to NK cells. These results indicate that NCT cells, which have different characteristics from NK cells, are present in normal, nonimmunized mouse spleen cells. Unexpectedly, CD4+ T cells sorted from normal mouse spleen T cells revealed significant NCT activity, as did CD8+ T cells. It was also demonstrated that NCT cells require the LFA-1 molecule to lyse tumor cells in the presence of anti-CD3 mAb.

T helper 2 (Th2) but not Th1 clones costimulate resting T cells in the presence of anti-CD3 monoclonal antibody

We have investigated the effects of monoclonal antibody (mAb) to the CD3 epsilon protein on interactions between small, resting T cells and antigen-specific T helper clones. Highly purified, splenic T cells lacking identifiable accessory cells do not proliferate in a thymidine uptake assay to anti-CD3 mAb, Con A, rIL-2, rIL-4, or irradiated T helper clones (both Th1 and Th2). However, the responding T cells proliferate significantly to the combined stimulus of Th2 clones and anti-CD3 antibody. Only the Th2, not the Th1, subpopulation of T helper cells has the ability to induce a T cell response. The Th2 cell-dependent activation of small resting T cells does not require the external cross-linkage of the anti-CD3 mAb via Fc receptor expressing cells or the secretion of lymphokines from the Th2 helper clones, but it is inhibitable by anti-LFA 1 antibody. Thus, Th2 clones provide a co-stimulatory signal which in conjunction with anti-CD3 mAb causes resting T cell proliferation in the absence of conventional accessory cells.

CD8 + T cells lyse autologous monocytes in the presence of anti-CD3 monoclonal antibody: Association with interleukin-1 production

Human peripheral blood T cells lysed autologous or allogeneic monocytes, but not polymorphonuclear cells (PMN) or lymphocytes, in the presence of anti-CD3 (OKT3 (IgG 2a) or anti-Leu 4 (IgG 1)) monoclonal antibody (mAb). Other mAbs such as OKT4 (IgG 2b), OKT8 (IgG 2a), OKT11 (IgG 2a), and OKM1 (IgG 2a) did not mediate lysis of monocytes. Lysis of monocytes also did not occur in the presence of F(ab′) 2 fragments of OKT3 mAb. OKT3 mAb and control murine IgG 2a mAb, but not F(ab′) 2 fragments of OKT3 mAb, were bound to the monocyte cell surface. Purified human IgG 1 and IgG 3 myeloma proteins, polyclonal human IgG, or Con A inhibited anti-CD3-dependent T-cell cytotoxicity against monocytes when added to the 4-hr 51Cr-release assay. Pretreatment of monocytes with an irrelevant murine IgG 2a mAb also inhibited OKT3 mAb (IgG 2a)-dependent lysis of these cells, but did not affect anti-Leu 4 mAb (IgG 1)-dependent lysis, suggesting that two different Fc receptors were involved. These results strongly suggest that Fc IgG receptors on monocytes are a critical structure for anti-CD3-dependent cytotoxicity. Lysis of monocytes was accompanied by interleukin-1 (IL-1) production, which was detected in supernatants from 4-hr cultures of T cells and monocytes in the presence of the OKT3 mAb. Both anti-CD3-dependent lysis of monocytes and IL-1 production were severely decreased after treatment of T cells with either OKT3 or OKT8 mAb plus complement, but were not affected significantly by treatment with the OKT4 mAb plus complement. Purified CD8 + cells, prepared using the cell sorter, exhibited significant levels of anti-CD3-dependent monocyte lysis (>10%). In contrast, purified CD4 + cells did not exhibit significant levels of anti-CD3-dependent cytotoxicity (<10%). Production of high concentrations of IL-1 was observed in cultures of purified CD8 + cells and monocytes in the presence of anti-CD3 mAb. Only low concentrations of IL-1 were detected in cultures of purified CD4 + cells, monocytes, and OKT3 mAb. These results suggest that CD8 + cells are primarily responsible for lysis of monocytes, which is associated with IL-1 production. It appears that anti-CD3 mAb brings CD8 + T cells and monocytes into close proximity by binding to the CD3 antigen on T cells and to the Fc IgG receptor on monocytes. This interaction results in lysis of monocytes primarily by CD8 + cells, after bypassing any antigen recognition requirements that may be otherwise needed. Lysis of monocytes appears to be associated with IL-1 release.

Characterization of murine thymocytes with CDS-associated T-cell receptor structures

The thymus is the major site for T-cell receptor (TCR) gene rearrangement and T-cell maturation. The specific antigen recognition structure (TCR) on murine T cells has been shown to be dependent on a polymorphic set of disulphide-linked heterodimers, containing two integral membrane glycoprotein chains, TCR alpha and TCR beta, expressed in non-covalent association with an invariant complex of proteins, CD3 (T3). Recently, a novel TCR/CD3 complex, that includes the product of the TCR gamma gene, has been identified on a subset of both peripheral cells and thymocytes. Here we examine the expression of TCR/CD3 complexes in fetal ontogeny and in the adult thymus. The results demonstrate that CD3+4-8-(T3+,L3T4-,Lyt2-)cells are detected in day-15 fetal thymi, throughout fetal development and in adult thymus. In situ hybridization studies indicate that these early CD3+ cells express high levels of TCR gamma-specific RNA, low levels of TCR beta-specific RNA and no detectable TCR alpha-specific RNA. Day-16 CD3+,4-,8- fetal thymocytes can be activated to proliferate and demonstrate cytolytic activity when cultured in the presence of anti-CD3 monoclonal antibodies and interleukin-2 (IL-2). These results suggest that CD3-bearing cells, present early in thymic ontogeny, express a functional TCR and may, therefore, be important in repertoire development.