Induced Peripheral Blood Mononuclear Cells Proliferation Research Articles

Potential SLA Hp-4.0 haplotype-restricted CTL epitopes identified from the membrane protein of PRRSV induce cell immune responses.

Swine leukocyte antigen (SLA) class I molecule-restricted T-cell epitopes, which induce cytotoxic T lymphocyte (CTL) responses, play a critical role in the clearance of porcine reproductive and respiratory syndrome virus (PRRSV) and the development of efficient protective vaccines. The SLA-1*04:01:01, SLA-2*04:01, and SLA-3*04:01 alleles, assigned the Hp-4.0 haplotype, are highly prevalent and usually present in all pig breeds. However, the SLA Hp-4.0 haplotype-restricted CTL epitopes in the structural membrane (M) protein of PRRSV are still unknown. In this study, we predicted 27 possible 9-mer epitope peptides in M protein with high binding scores for SLA-1*04:01:01 using CTL epitope prediction tools. In total, 45 SLA class I complexes, comprising the predicted peptide, extracellular region of the SLA-I molecules, and β2-microglobulin, were constructed in vitro to detect the specific binding of these peptides to SLA-1*04:01:01 (27 complexes), SLA-2*04:01 (9 complexes), and SLA-3*04:01 (9 complexes), respectively. Our results showed that the M27 (T91WKFITSRC), M39 (N130HAFVVRRP), and M49 (G158RKAVKQGV) peptides bind specifically to SLA-1*04:01:01, SLA-2*04:01, and SLA-3*04:01, respectively. Subsequently, using peripheral blood mononuclear cells (PBMCs) isolated from the homozygous Hp-4.0 and Hp-26.0 haplotype piglets vaccinated with commercial PRRSV HuN4-F112 strain, we determined the capacities of these 27 potential peptides to stimulate their proliferation with a Cell Counting Kit-8 and their secretion and expression of interferon gamma (IFN-γ) with an ELISpot assay and real-time qPCR, respectively. The immunological activities of M27, M39, and M49 were therefore confirmed when they efficiently induced PBMC proliferation and IFN-γ secretion in PBMCs from piglets with the prevalent SLA Hp-4.0 haplotype. The amino acid sequence alignment revealed that M27, M39, and M49 are highly conserved among 248 genotype II PRRSV strains collected between 1998 and 2019. These findings contribute to the understanding of the mechanisms of cell-mediated immune responses to PRRSV. Our study also provides a novel strategy for identifying and confirming potential SLA haplotype-restricted CTL epitopes that could be used to develop novel peptide-based vaccines against swine diseases.

Anti-inflammatory properties of an isoxazole derivative - MZO-2.

A series of new isoxazole derivatives of expected immunosuppressive activities was synthesized. Following in vitro screening in the human cell models, the activity of MZO-2 compound (ethyl N-{4-[(2,4-dimethoxybenzyl)carbamoyl]-3-methylisoxazol-5-yl}acetimidate) in mouse in vivo models was evaluated. In vitro tests included evaluation of: peripheral blood mononuclear cells (PBMC) viability, phytohemagglutinin (PHA)-induced PBMC proliferation and lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNF α) production in whole blood cell cultures. MZO-2 was studied in mice for its effects on: humoral immune response to sheep erythrocytes (SRBC), delayed type hypersensitivity (DTH) to ovalbumin (OVA), contact sensitivity to oxazolone and carrageenan-induced foot pad edema. In addition, the effect of MZO-2 on expression of caspases in Jurkat cells was determined. The studied compounds exhibited differential, dose-dependent effects to suppress PHA-induced PBMC proliferation and a weak property to suppress LPS-induced production of TNF α. MZO-2 had no effect on the induction phase of the humoral immune response to SRBC in vitro and in vivo, but moderately suppressed the induction phase of DTH to OVA. Its inhibitory effect on carrageenan-induced paw inflammation was potent. Likewise, MZO-2, applied in ointment, was very effective in reducing ear edema and number of lymphocytes in draining lymph nodes of mice sensitized to oxazolone, comparably to tacrolimus, the reference drug. The expression of caspases 3, 8 and 9 in Jurkat cells was inhibited by the compound. MZO-2, applied systemically or locally, may serve as a potential drug for amelioration of inflammatory processes.

Truncated Escherichia coli thioredoxin induces proliferation of human blood mononuclear cells and production of reactive oxygen species as well as proinflammatory cytokines.

Thioredoxin (Trx) is a redox protein characterized by a Trx fold. A naturally occurring truncated human Trx, Trx 80, which lacks the C-terminal strand-helix of the Trx fold, stimulates proliferation of peripheral blood mononuclear cells (PBMCs). It has not been clear how Trx80 gains this function. This study investigates whether a peptide with substantial sequence difference from Trx80, but retaining an abridged Trx fold can elicit PBMC proliferation. We genetically truncated a carboxy-terminal β-α motif of Escherichia coli Trx to produce a peptide, Trx83, which shares low sequence identity with human Trx80. Addition of reduced-form Trx83 to resting human PBMCs promoted cell proliferation, while oxidized-form Trx83 lacked the function. By contrast, oxidized-form Trx80 exhibited a high activity in promoting PBMC proliferation, indicating the importance of sequence context of an abridged thioredoxin in influencing PBMC proliferation. Trx83 increases cellular reactive oxygen species and proinflammatory cytokines TNF-α and IL-1β, suggesting that Trx83 modulates inflammatory pathways. This notion is supported by the observation that cystine or cysteine abolishes the Trx83 induced PBMC proliferation. The PBMC stimulatory activity of Trx83 may have potential for pharmacological developments. Elicitation of primary proliferative responses of PBMCs by a protein is generally difficult. We show that Escherichia coli Trx83 with a truncated Trx fold induces PBMC proliferation, but only in the disulfide-reduced form. In contrast, oxidized-form human Trx80 is a potent stimulator. These results demonstrate that the sequence context of an abridged Trx fold is influential in inducing PBMC proliferation. The stimulatory effect of Trx83 is associated with an increase of inflammatory response. The possibility of eliciting PBMC proliferation and switching this activity on/off by redox control provides a perspective for developing Trx83 as a PBMC stimulatory agent. Copyright © 2016 John Wiley & Sons, Ltd.

THU0175 T cell reactivity to β2 glycoprotein I in systemic lupus erythematosus and anti-phospholipid syndrome: Correlation with subclinical atherosclerosis

BackgroundSystemic lupus erythematosus (SLE) and anti-phospholipid syndrome (APS) patients present a high prevalence of atherosclerosis associated with both traditional and disease-related risk factors. β2 glycoprotein I (β2GPI), the main antigen...

Immunogenicity and Immunomodulatory Properties of Hepatocyte-like Cells Derived from Human Amniotic Epithelial Cells

Hepatocyte transplantation is being trialled as an alternative to whole organ transplant for patients with acute liver failure and liver specific metabolic diseases. Due to the scarcity of human hepatocytes, hepatocyte-like cells (HLC) generated from stem cells may become a viable alternative to hepatocyte transplantation. Human amniotic epithelial cells (hAEC) from the placenta have stem cell-like properties and can be differentiated into HLC. Naïve hAEC have low immunogenicity and exert immunomodulatory effects that may facilitate allogeneic transplantation. However, whether the immunogenicity and immunomodulatory properties alter with differentiation into HLC are unknown. We further characterized HLC generated from hAEC, examined changes in human leucocyte antigens (HLA) and co-stimulatory molecules and effects exerted by the HLC on human peripheral blood mononuclear cells (PBMC). HLC derived from hAEC expressed proteins found in hepatocytes, had CYP3A4 drug metabolizing enzyme activity and secreted urea. IFN-γ treatment increased HLA Class IA, Class II and co-stimulatory molecule CD40 expression in the HLC. IFN-γ treated HLC stimulated proliferation of PBMC in one-way mixed lymphocyte reactions and were more immunogenic than undifferentiated hAEC. However, the HLC showed immunomodulatory properties and inhibited mitogen induced PBMC proliferation in vitro. PBMC proliferation may have been inhibited by IL-6, TGF-β1, PGE2 and HLA-G secreted by the HLC. The retention of immunomodulatory properties may enable HLC grafts to survive for longer periods despite the immunogenicity of the HLC.

In silico prediction of Ara h 2 T cell epitopes in peanut‐allergic children

Despite the frequency and severity of peanut allergy, the only approved treatment is strict avoidance. Different types of immunotherapy with crude peanut extract are not universally effective and have been associated with relatively high adverse reaction rates. We sought to determine whether in silico predictive algorithms were useful in identifying candidate peptides for an Ara h 2 peptide-based vaccine using peanut-allergic patients' peripheral blood mononuclear cells (PBMCs) in vitro. A human leucocyte antigen (HLA) distribution analysis was also performed. Major histocompatibility complex (MHC)-class II-binding peptides were predicted using NetMHCIIpan-2.0 and NetMHCII-2.2 algorithms. PBMCs from 80 peanut-allergic patients were stimulated with overlapping 20-mer Ara h 2 peptides. Cell supernatant cytokine profiles were evaluated by multiplex assays. HLA-DRB1* and HLA-DQB1* typing were performed. Four regions of overlapping sequences induced PBMC proliferation and predominant Th2 cytokine production. HLA genotyping showed 30 different DRB1* allele specificities and eight DQ serological specificities. The in silico analysis revealed similar relevant regions and predicted identical or similar core 9-mer epitopes to those identified in vitro. If relevant peptides, as determined by either in vitro or in silico analysis (15 peptides and 9 core epitopes respectively), were used in a peptide-based vaccine, they would cover virtually all subjects in the cohort studied. Four dominant regions in Ara h 2 have been identified, containing sequences that could serve as potential candidates for peptide-based immunotherapy. MHC-class II-based T cell epitope prediction algorithms for HLA-DR and -DQ loci accurately predicted Ara h 2 T cell epitopes in peanut-allergic subjects, suggesting their potential utility in a peptide-based vaccine design for food allergy.

Soluble CD26 / Dipeptidyl Peptidase IV Enhances Human Lymphocyte Proliferation In Vitro Independent of Dipeptidyl Peptidase Enzyme Activity and Adenosine Deaminase Binding

Human CD26 has dipeptidyl peptidase-4 (DPP IV) enzyme activity and binds to adenosine deaminase (ADA). CD26 is costimulatory for lymphocytes and has a circulating soluble form (sCD26). DPP IV enzyme inhibition is a new successful type 2 diabetes therapy. We examined whether the ADA binding and catalytic functions of sCD26 contribute to its effects on T-cell proliferation. Wildtype soluble recombinant human CD26 (srhCD26), an enzyme inactive mutant (srhCD26E-) and an ADA non-binding mutant (srhCD26A-) were co-incubated in in vitro T-cell proliferation assays with peripheral blood mononuclear cells (PBMC) stimulated with phytohaemagglutinin (PHA), muromonab-CD3 or Herpes simplex virus antigen (HSV Ag). Both srhCD26 and srhCD26E- enhanced PHA-induced T-cell proliferation dose-dependently in all six subjects tested. srhCD26 and srhCD26A- had no overall effect on anti-CD3-stimulated PBMC proliferation in four of five subjects. srhCD26, srhCD26E- and srhCD26A- enhanced HSV Ag induced PBMC proliferation in low responders to HSV Ag, but had no effect or inhibited proliferation in HSV-high responders. Thus, effects of soluble human CD26 on human T-cell proliferation are mechanistically independent of both the enzyme activity and the ADA-binding capability of sCD26.

Reduced CD4 +T cell activation in children with type 1 diabetes carrying the PTPN22/Lyp 620Trp variant

The 620Trp variant of the LYP protein, encoded by the lymphoid tyrosine phosphatase 22 gene ( PTPN22), is associated with autoimmunity. In this study we aimed at characterising the role of this variant on lymphocyte activation. We analysed cytokine secretion and proliferation of peripheral blood mononuclear cells (PBMCs) and CD4 +T cells in a cohort of clinically non-diabetic, multiple autoantibody-positive children, healthy controls and in children with type 1 diabetes (T1D). We found a decreased proliferation and IL-2 production of CD4 +T cells after anti-CD3/anti-CD28 stimulation ( p = 0.04 for IL-2) among T1D patients. In addition, a profoundly decreased intracellular calcium flux in CD4 +T cells after PHA stimulus was detected among 620Trp carriers. In contrast, no effect of this polymorphism on tuberculin and tetanus toxoid induced PBMC proliferation and cytokine secretion was observed in autoantibody positive children, healthy controls and children with newly-diagnosed T1D. In conclusion, the LYP 620Trp variant is associated with reduced activation, proliferation and IL-2 production in CD4 +T cells among T1D patients. In accordance with our previous findings on the key role of this variant on disease progression, this mechanism is likely to contribute to the development of β-cell specific autoimmunity.

Biological characterization of glutaraldehyde-modified Parietaria judaica pollen extracts.

Allergoids are widely used in specific immunotherapy (SIT) for the treatment of IgE-mediated allergic diseases, but all techniques for standardization of conventional allergic extracts may not be appropriate for standardization of a glutaraldehyde (GA)-modified extract because of the unique characteristics of these extracts. To assess an accurate methodology for standardization of chemically modified extracts. GA-modified extracts from Parietaria judaica pollen were purified by diafiltration. Biochemical properties were investigated by determination of amino groups, chromatography, and SDS-PAGE. The IgE-binding activity was determined by skin prick test, enzyme allergosorbent test inhibition, basophil activation, and histamine release tests. Peripheral blood mononuclear cells (PBMCs) from P. judaica pollen-allergic subjects were stimulated with either native or allergoid extracts, and proliferation was measured. Biochemical data indicated a high degree of allergen polymerization resulting in extract components higher than 100 kDa. IgE-binding activity, both in vivo and in vitro, was reduced by more than 99.8%. Both allergen and allergoid induced PBMC proliferation and synthesis of blocking IgG antibodies at similar rates. Moreover, no evidence of introduction of new determinants by chemical modification was found. The preparation of GA-modified extracts by diafiltration is faster and more reliable than previous chromatographic methods. These modified extracts have drastically reduced their allergenicity while maintaining their immunogenicity, and therefore they can be used in safer and shortened schedules of SIT.

Patients on Chronic Hemodialysis Have No Intrinsic Lymphocyte Defect upon Stimulation with Interleukin-2, Interleukin-15 or Tumor Necrosis Factor-Alpha

Background: Patients on chronic hemodialysis (HD) suffer from general immune incompetence, resulting in a high incidence of infectious complications, impaired response to vaccinations and a high incidence of malignancy. Although various abnormalities in T cell function of HD patients have been described, it remains unclear whether this is due to an intrinsic T cell defect. Aim: In the present study we tested the capacity of T cells to proliferate upon stimulation with antigen-presenting cell and T-cell-derived cytokines. Methods: The proliferation capacity of lymphocytes obtained from patients on HD and healthy controls was determined by measuring the proliferation of peripheral blood mononuclear cells (PBMC) after stimulation with rhIL-2, rhIL-15, rhTNF-α, or combination of those cytokines. In all samples the percentage of α/β TCR-positive T cells was measured. Results: After isolation of PBMC the percentage of T cells varied from 70% (before stimulation) to 80% (after stimulation). IL-2, IL-15 and TNF-α all induced PBMC proliferation, while the combination TNF-α plus IL-2 or TNF-α plus IL-15 appeared to be additive. No difference between PBMC from HD patients and controls was found. Conclusion: We conclude that lymphocytes from HD patients have no intrinsic defects in their proliferation capacity after stimulation with IL-2, IL-15 or TNF-α, in vitro, as the increase in counts per minute is predominant.

Cytokine production in nickel-sensitized individuals analysed with enzyme-linked immunospot assay: possible implication for diagnosis.

Patients with suspected allergic contact dermatitis still have to undergo patch testing for a correct diagnosis. As this has several disadvantages there is a need for additional methods, preferentially those that can be performed in vitro. Objectives To investigate the possibility of diagnosing contact allergy to nickel (Ni2+) using the enzyme-linked immunospot (ELISpot) assay that allows the analysis of cytokines at a single-cell level in ex vivo activated peripheral blood mononuclear cells (PBMC). Eleven female patients and nine age- and sex-matched healthy volunteers participated in the study. All patients had a history of nickel allergy and a positive patch test reaction to NiSO4, while the controls' test was negative. PBMC were cultured in the presence or absence of NiCl2. Cell proliferation was measured with [3H]thymidine incorporation, and the number of cytokine-producing cells analysed with the ELISpot assay. The proliferative response of PBMC to Ni2+, expressed as stimulation index, was significantly higher in the nickel-allergic patients than in the control group. Using the ELISpot assay, we found that PBMC from nickel-allergic individuals responded to Ni2+ with significantly greater production of interleukin (IL)-4, IL-5, IL-13 and interferon-gamma, but not IL-12, compared with the healthy controls. The number of IL-4- and IL-5-producing cells correlated with the number of IL-13-producing cells in the nickel-allergic patients, but Ni2+-induced PBMC proliferation did not correlate with the number of cytokine-producing cells for any of the cytokines tested. Our results indicate that the ELISpot assay could be a tool in the discrimination between nickel-allergic and non-allergic individuals.

Determination of peripheral blood mononuclear cell responses to mitogens and woodchuck hepatitis virus core antigen in woodchucks by 5-bromo-2'-deoxyuridine or 2[3H]adenine incorporation.

Characterization of cellular immune response to antigens of woodchuck hepatitis virus (WHV) can contribute to the understanding of acute resolving and chronic outcome of hepadnavirus infection. Studies were limited because peripheral blood mononuclear cells (PBMCs) of woodchucks failed to incorporate [3H]thymidine sufficiently. Therefore, we established a non-radioactive proliferation assay for woodchuck PBMCs using 5-bromo-2'-deoxyuridine (BrdU) as thymidine analogue. Mitogen- and WHV core protein-(WHcAg) induced PBMC proliferation was detected by BrdU incorporation and compared to an assay using 2[3H]adenine. After stimulation with concanavalin A (ConA) and phytohaemagglutinin (PHA) we observed significant PBMC proliferation with both assays. Mitogen-induced nucleoside uptake of PBMCs into cellular DNA was confirmed by detection of 1',2'[3H]BrdU and 2[3H]adenine in extracted DNA. PBMCs obtained during the acute phase of WHV infection could be stimulated by WHcAg, whereas no WHcAg-induced proliferation of PBMCs was found in WHV-negative animals. PBMCs of chronic WHV carriers showed only a weak response to WHcAg. The established assays will be useful in determining the kinetics of cellular immune responses to different WHV antigens in the course of WHV infection and may provide an insight into mechanisms responsible for chronic outcome of hepadnavirus infection.

HIV-1 epitopes exposed by hybrid hepatitis B core particles affect proliferation of peripheral blood mononuclear cells from HIV-1 positive donors

Hepatitis B virus core protein (HBc) in particulate form is a potent immunogen for the design of vaccines with a broad T-cell reactivity. Hybrid HBc proteins with N-terminal insertions of human immunodeficiency virus (HIV-1) B- and T-cell epitopes from gp41 and p34 pol, respectively, were constructed. HBc hybrids formed particles with HIV-1 epitopes exposed on the surface. The proliferative response of peripheral blood mononuclear cells (PBMC) from HIV-1 infected donors to the hybrids was studied in vitro and compared to that of synthetic peptides representing the same HIV-1 sequences. The epitope from p34 pol induced PBMC proliferation both when inserted into HBc and as a peptide. The epitope from gp41, when inserted into HBc, and to a lesser extent the relevant peptide caused a decreased reactivity. The inhibitory effect of the HBc hybrid carrying the gp41-epitope was pronounced even in the HIV-1 infected donors with proliferative responses to HBc. The similarity between the mode of action of the peptides and hybrid HBcs implies either correct processing of the latter or T-cell recognition of HIV-1 epitopes in the intact hybrid HBc particles.

Quantification of mitogen induced human lymphocyte proliferation: Comparison of alamarbluetm assay to 3h‐thymidine incorporation assay

Proliferation of human lymphocytes in response to various stimuli has traditionally been assessed by measuring uptake of radiolabeled nucleotides such as 3H-thymidine. We have evaluated a fluorometric assay, which uses the commercially available reagent, alamarBlue, as a potential substitute for the 3H-thymidine assay in measuring proliferation of human lymphocytes. In this assay, alamarBlue is added to a population of cells where it is reduced by mitochondrial enzyme activity. The reduced form of the reagent is fluorescent and can be quantitatively detected. The safety and convenience of the alamarBlue assay make it very attractive for use in the clinical laboratory. In this study peripheral blood mononuclear cells (PBMC) from healthy donors were stimulated using the mitogen Concanavalin A, and proliferation was assessed using either the 3H-thymidine or the alamarBlue assay. The alamarBlue assay reliably detects human PBMC and we found that the linear range of detection was 10(4) cells/well (96-well plate) to 5 x 10(5) cells/well. Detection of human PBMC is highly reproducible and the alamarBlue assay may be suitable in a number of applications where detection or relative quantitation of human PBMC is required. The alamarBlue assay also detected mitogen induced proliferation of PBMC although with a significantly lower level of sensitivity than the standard 3H-thymidine assay.

Role of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) in peripheral blood mononuclear cell activation by human renal carcinoma cells.

We examined the role of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) during the activation of peripheral blood mononuclear cells (PBMCs) by the human renal carcinoma cell line CaKi-1. ICAM-1 antigen expression was induced on CaKi-1 cells by incubation with either phorbol-12-myristate 13 acetate (PMA) or interferon-gamma (IFN-gamma). Following a thorough washout of PMA and IFN-gamma and subsequent paraformaldehyde fixation, CaKi-1 cell monolayers were cocultered with allogenic PBMCs. While PMA-treated CaKi-1 cells induced PBMC proliferation and interleukin-2 receptor antigen expression, this was not the case for control or IFN-gamma-treated CaKi-1 cells. Furthermore, the induced PBMC proliferation was inhibited by specific monoclonal antibodies against ICAM-1 and LFA-1. Finally, although PMA induced human leukocyte antigen (HL)-A, B, C antigen expression on CaKi-1 cells, a monoclonal antibody against this antigen did not inhibit PBMC proliferation. We conclude that PMA can modulate CaKi-1 cells to stimulate allogenic PBMC proliferation in an ICAM-1/LFA-1 dependent, but HLA-A, B, C-independent, fashion. This stimulation might reside in the long-term activation of protein kinase C, induced by PMA.

Mapping of T cell epitopes of the major fraction of rye grass using peripheral blood mononuclear cells from atopics and non-atopics. II. Isoallergen clone 5A of Lolium perenne group I (Lol p I).

Rye grass is the major cause of hay fever which currently affects 20% of the population. Lolium perenne group I (Lol p I) is a glycoprotein of 240 amino acid residues, representing the main allergen of rye grass. We have used peripheral blood mononuclear cells (PBMC) from controls and subjects allergic to rye grass and cultured them with L. perenne extract (LPE) and Lol p I and measured lymphocyte activation using thymidine incorporation. Patients were further studied against the 115 overlapping peptides of the iso-allergen clone 5A of Lol p I to see whether the 4 amino acid residue differences between clone 1A and clone 5A affect the T cell epitope and thus, lymphocyte activation. There are 24 peptide differences between isoallergen clone 1A and clone 5A occurring in pools 4, 13, 16 and 19 each one of which could be an immunodominant epitope. The PBMC from all allergic patients studied showed a strong proliferative response to LPE and Lol p I. Five immunogenic peptide pools, pool 6, 15, 16, 17 and 19 of the isoallergen clone 5A were also identified. Most of these pools are in the C-terminal region of Lol p I. Out of 20 pools tested in vitro 1 pool (pool-17) induced PBMC proliferation in five out of six patients who were not restricted to an HLA class II DR gene product. However, three out of the six subjects responded to various other peptide pools in addition to the immunodominant pool. In spite of the amino acid differences between the two clones, pool 17 still remains the immunodominant T cell epitope. Control subjects showed only weak responses to LPE and no detectable response to either Lol p I or peptide pools. From within the most active pool we have defined two peptides of the isoallergen clone 5A (identical in sequence with clone 1A) which stimulate lymphocytes from rye grass-sensitive patients in vitro. Previous studies with the two continuous sequences (193WGAVWRIDTPDK204 and 195AVWRIDTPDKLT206) tested in vivo by intradermal skin testing have shown typical delayed-type hypersensitivity reactions after 24-48 h in one patient. Comparison of amino acid sequences of Lol p I, Lol p II and Lol p III proteins revealed a significant level of structural similarity among them. Interestingly, 50% of the residues of the second peptide sequence are also present in Lol p II and Lol p III.(ABSTRACT TRUNCATED AT 400 WORDS)

A Monoclonal Antibody (H227) Recognizing a New Epitope of 4F2 Molecular Complex Associated with T Cell Activation

We developed and characterized a monoclonal antibody (mAb), H227, recognizing a new epitope of human 4F2 molecular complex whose roles remain to be identified. Both staining patterns and molecular sizes recognized by H227 mAb were the same to those of 4F2 mAb. Thus, both H227 and 4F2 mAbs were reactive to monocytes, thymocytes, and activated lymphocytes and immunoprecipitated the 125-kDa molecular weight membrane protein under nonreducing conditions and 85-kDa heavy-chain and 40-kDa light-chain proteins under reducing conditions. These two mAbs immunoprecipitated only the 85-kDa heavy-chain protein when the cell lysate was initially treated with dithiothreitol. Sequential immunoprecipitation proved their cross-reactivity. Both the mAbs inhibited phytohemagglutinin- or concanavalin A-induced proliferation of peripheral blood mononuclear cells (PBMC). In contrast to these similarities, the pretreatment of cells with one mAb failed to block the reactivity to the other, suggesting that their epitopes were different from each other. Furthermore, only H227 mAb augmented phorbol myrislate acetate (PMA)-induced PBMC proliferation in association with increase in interleukin 2 receptor expression. In summary, H227 mAb recognizes a new epitope of 4F2 heavy chain that might be involved in the PMA-induced T cell activation pathway and therefore shall be a useful tool for understanding the roles of the 4F2 molecular complex.