Unstimulated Ones Research Articles

Transcriptomic study reveals alteration in the expression of long non-coding RNAs (lncRNAs) during reversal of HIV-1 latency in monocytic cell line.

The presence of latent HIV reservoirs continues to be the biggest obstacle to achieving an HIV cure. Thus, long non-coding RNAs (lncRNAs) may serve as the preferred targets for HIV latency reversal. The goal of the study was to identify prospective lncRNAs for subsequent in vitro molecular and functional characterization. RNA-sequencing was performed in latently HIV-infected monocytic cell line (U1) under stimulated and unstimulated condition using Illumina-HiSeqX platform, followed by its validation using qRT-PCR assay. Gene ontology (GO), KEGG pathway, and co-expression analyses were performed to identify the enriched biological processes and pathways in U1 cells post-stimulation with the latency reversal agent SAHA. A total of 3,576 and 1,467 significantly altered lncRNAs and protein-coding genes respectively, were identified in SAHA-stimulated U1 cells compared to unstimulated ones. The GO and KEGG pathway analyses of the differentially expressed protein-coding genes showed the enrichment of diverse biological processes and pathways respectively, in SAHA-stimulated U1 cells. Co-expression analysis between lncRNAs and protein-coding gene pairs, helped predict potential pathways with which these lncRNAs are associated. Further in vitro validation in HIV-infected monocytes showed that the expression of the top two candidate lncRNAs, LINC01231 and LINC00560, are specific to HIV infection. Transcriptome analysis revealed changes in the expression of numerous lncRNAs and protein-coding genes following stimulation with SAHA. Co-expression analysis identified candidate lncRNAs and their associated biological pathways. However, additional in vitro experimental exploration using gene knockdown strategies is needed to ascertain the specific role of LINC01231 and LINC00560 lncRNAs in latently infected monocytes.

Dental Pulp Stem Cells Modulate Inflammasome Pathway and Collagen Deposition of Dermal Fibroblasts.

Fibrosis is a pathological condition consisting of a delayed deposition and remodeling of the extracellular matrix (ECM) by fibroblasts. This deregulation is mostly triggered by a chronic stimulus mediated by pro-inflammatory cytokines, such as TNF-α and IL-1, which activate fibroblasts. Due to their anti-inflammatory and immunosuppressive potential, dental pulp stem cells (DPSCs) could affect fibrotic processes. This study aims to clarify if DPSCs can affect fibroblast activation and modulate collagen deposition. We set up a transwell co-culture system, where DPSCs were seeded above the monolayer of fibroblasts and stimulated with LPS or a combination of TNF-α and IL-1β and quantified a set of genes involved in inflammasome activation or ECM deposition. Cytokines-stimulated co-cultured fibroblasts, compared to unstimulated ones, showed a significant increase in the expression of IL-1β, IL-6, NAIP, AIM2, CASP1, FN1, and TGF-β genes. At the protein level, IL-1β and IL-6 release as well as FN1 were increased in stimulated, co-cultured fibroblasts. Moreover, we found a significant increase of MMP-9 production, suggesting a role of DPSCs in ECM remodeling. Our data seem to suggest a crosstalk between cultured fibroblasts and DPSCs, which seems to modulate genes involved in inflammasome activation, ECM deposition, wound healing, and fibrosis.

Novel RNA biomarkers improve discrimination of children with tuberculosis disease from those with non-TB pneumonia after in vitro stimulation.

The diagnosis of pediatric tuberculosis (TB) poses a challenge for clinical teams worldwide. TB-mediated changes in the expression of host genes in the peripheral blood can serve as diagnostic biomarkers and can provide better insights into the host immune mechanisms of childhood TB. Peripheral blood mononuclear cells (PBMCs) from children (n=102) with microbiologically confirmed TB disease, TB infection (TBI), pneumonia, and healthy controls (HC) were stimulated with either the Purified Protein Derivative (PPD) or the Early Secretory Antigen 6kDa-Culture Filtrate Protein 10 (ESAT6-CFP10) complex of Mycobacterium tuberculosis (Mtb). RNA was extracted and quantified using gene expression microarrays. Differential expression analysis was performed comparing microbiologically confirmed TB to the other diagnostic groups for the stimulated and unstimulated samples. Using variable selection, we identified sparse diagnostic gene signatures; one gene (PID1) was able to distinguish TB from pneumonia after ESAT6-CFP10 stimulation with an AUC of 100% in the test set, while a combination of two genes (STAT1 and IFI44) achieved an AUC of 91.7% (CI95% 75.0%-100%) in the test set after PPD stimulation. The number of significantly differentially expressed (SDE) genes was higher when contrasting TB to pneumonia or HC in stimulated samples, compared to unstimulated ones, leading to a larger pool of candidate diagnostic biomarkers. Our approach provides enlightened aspects of peripheral TB-specific responses and can form the basis for a point of care test meeting the World Health Organization (WHO) Target Product Profile (TPP) for pediatric TB.

Immune Responses in Cutaneous Leishmaniasis: In vitro Thelper1/Thelper2 Cytokine Profiles Using Live Versus Killed Leishmania major.

Background:Recovery from cutaneous leishmaniasis (CL) leads to protection against further lesion development. In contrast, vaccination using killed parasites does not induce enough protection; the reason(s) is not currently known but might be related to different immune response induced against live versus killed parasites. In this study, Th1/Th2 cytokine profiles of CL patients were evaluated against live versus killed Leishmania major.Methods:In this study peripheral blood mononuclear cells (PBMC) of the volunteers with active CL lesion (CL), history of CL (HCL) and healthy volunteers were cultured and stimulated with live or killed Leishmania major, the supernatants were collected and levels of IFN-γ, IL-5 and IL-10 were titrated using ELISA method.Results:The results showed that IFN-γ levels in CL patients (p< 0.001) and HCL volunteers (p< 0.005) are significantly higher when stimulated with live than stimulated with killed L. major. IFN-γ production in PBMC volunteers with CL and HCL stimulated with live or heat-killed L. major was significantly (p< 0.001) higher than in unstimulated ones. The level of IL-5 in CL patients (p< 0.005) and HCL volunteers (p< 0.001) are significantly lower when stimulated with live than killed L. major. There was no significant difference between the levels of IL-10 in PBMC stimulated with either live or killed L. major.Conclusion:It is concluded that using live Leishmania induces a stronger Th1 type of immune response which justify using leishmanization as a control measure against CL.

Performance of anti-CD19 chimeric antigen receptor T cells in genetically defined classes of chronic lymphocytic leukemia

BackgroundWhile achieving prolonged remissions in other B cell-derived malignancies, chimeric antigen receptor (CAR) T cells still underperform when injected into patients with chronic lymphocytic leukemia (CLL). We studied the influence...

Human odontoblast-like cells produce nitric oxide with antibacterial activity upon TLR2 activation.

The penetration of cariogenic oral bacteria into enamel and dentin during the caries process triggers an immune/inflammatory response in the underlying pulp tissue, the reduction of which is considered a prerequisite to dentinogenesis-based pulp regeneration. If the role of odontoblasts in dentin formation is well known, their involvement in the antibacterial response of the dental pulp to cariogenic microorganisms has yet to be elucidated. Our aim here was to determine if odontoblasts produce nitric oxide (NO) with antibacterial activity upon activation of Toll-like receptor-2 (TLR2), a cell membrane receptor involved in the recognition of cariogenic Gram-positive bacteria. Human odontoblast-like cells differentiated from dental pulp explants were stimulated with the TLR2 synthetic agonist Pam2CSK4. We found that NOS1, NOS2, and NOS3 gene expression was increased in Pam2CSK4-stimulated odontoblast-like cells compared to unstimulated ones. NOS2 was the most up-regulated gene. NOS1 and NOS3 proteins were not detected in Pam2CSK4-stimulated or control cultures. NOS2 protein synthesis, NOS activity and NO extracellular release were all augmented in stimulated samples. Pam2CSK4-stimulated cell supernatants reduced Streptococcus mutans growth, an effect counteracted by the NOS inhibitor L-NAME. In vivo, the NOS2 gene was up-regulated in the inflamed pulp of carious teeth compared with healthy ones. NOS2 protein was immunolocalized in odontoblasts situated beneath the caries lesion but not in pulp cells from healthy teeth. These results suggest that odontoblasts may participate to the antimicrobial pulp response to dentin-invading Gram-positive bacteria through NOS2-mediated NO production. They might in this manner pave the way for accurate dental pulp healing and regeneration.

Human endotoxin tolerance is associated with enrichment of the CD14+ CD16+ monocyte subset

Prior exposure to lipopolysaccharides (LPS) induces a state of cell resistance to subsequent LPS restimulation, known as endotoxin tolerance, mainly by repressing the expression of pro-inflammatory cytokines. We established an endotoxin tolerance model in human monocytes Endotoxin-tolerant cells showed a decrease in IκBα degradation and diminished expression of Tumor necrosis factor (TNF) (both messenger RNA [mRNA] and protein content). The myeloid differentiation factor 88 (MyD88)/MyD88 splice variant (MyD88s) ratio, an indirect way to test the Toll-like receptor 4 (TLR4) MyD88-dependent signaling cascade, did not change in endotoxin-tolerant cells when compared to LPS-stimulated or -unstimulated ones. Remarkably, cell population analysis indicated a significant increase of the CD14+ CD16+ subset only under the endotoxin-tolerant condition. Furthermore, endotoxin-tolerant cells produced higher amounts of C–X–C motif chemokine 10 (CXCL10), a typical MyD88-independent cytokine.

Prenatal vitamin D₃ supplementation suppresses LL-37 peptide expression in ex vivo activated neonatal macrophages but not their killing capacity.

Vitamin D has regulatory effects on innate immunity. In the present study, we aimed to assess the effect of prenatal vitamin D₃ (vitD₃) supplementation on neonatal innate immunity in a randomised, placebo-controlled trial by evaluating cathelicidin (LL-37) expression and the killing capacity of macrophages. Healthy pregnant women (n 129) attending a clinic in Dhaka were randomised to receive either a weekly oral dose of 0·875 mg vitD₃ or placebo starting from 26 weeks of gestation up to delivery. Serum, plasma and monocyte-derived macrophages (MDM) were obtained from the cord blood. 25-Hydroxyvitamin D (25(OH)D) concentration was measured in serum. MDM were stimulated with or without Toll-like-receptor 4 ligand (TLR4L). Innate immune function was assessed by measuring LL-37 peptide levels in the culture supernatant of MDM by ELISA, LL-37 transcript levels by quantitative PCR, and ex vivo bactericidal capacity of MDM. VitD₃ supplementation did not increase LL-37 peptide levels in plasma or in the extracellular fluid of macrophages with or without TLR4L induction. However, stimulated intracellular LL-37 expression (ratio of stimulated:unstimulated MDM) was significantly reduced in the vitamin D group v. placebo (P=0·02). Multivariate-adjusted analyses showed that intracellular LL-37 peptide concentration from stimulated MDM was inversely associated with 25(OH)D concentration in serum (P=0·03). TLR4L stimulation increased the bactericidal capacity of MDM compared with the unstimulated ones (P=0·01); however, there was no difference in killing capacity between the two groups. A weekly dose of 0·875 mg vitD₃ to healthy pregnant women suppressed the intracellular LL-37 peptide stores of activated macrophages, but did not significantly affect the ex vivo bactericidal capacity of cord blood MDM.

Langerhans Cells Stimulated by Mechanical Stress Are Susceptible to Measles Virus Infection

Objective: Measles virus (MV) first infects the human respiratory tract, but the initial target cells are unknown. We examined whether MV infects Langerhans cell-like dendritic cells (LCs) generated from CD14⁺ monocytes in the presence of GM-CSF, IL-4, and TGF-β1. Methods: Cultured LCs were established as described recently [Biochem Biophys Res Commun 2003;306:674–679]. The expression of immunological markers was detected by FACScan. Infection with MV was assessed by syncytia formation, viral-specific fluorescence, and Western blotting. Results: MV did not infect and replicate the freshly established, unstimulated LCs expressing CD1a, E-cadherin and Langerin but not CD83. Also, CD150, a receptor for MV was not expressed on the surface of the LCs. However, LCs stimulated by mechanical stress such as washing and centrifugation became susceptible to MV infection. Conclusion: A subset of mechanically stimulated LCs but not unstimulated immature ones became susceptible to MV. The actual role of Langerhans cells in local immunity seems to be to suppress unfavorable reactions initiated by virus intrusion.

The role of cyclooxygenase inhibition in the effect of α-melanocyte-stimulating hormone on reactive oxygen species production by rat peritoneal neutrophils

The effect of α-MSH on reactive oxygen species (ROS) production by rat peritoneal neutrophils and the effect of cyclooxygenase (COX) inhibition were investigated using the chemiluminescence (CL) technique. Cells were obtained by peritoneal lavage 4 h after administration of oyster glycogen to rats and were stimulated with lipopolysaccharide (LPS) from Salmonella enderitidis and phorbol 12-myristate 13-acetate (PMA). The increasing concentrations of α-MSH (10 –12–10 –6 M) were added to stimulated cells alone or along with the COX inhibitors indomethacin, ketorolac or nimesulide (10 −8–10 −5 M). Luminol and lucigenin CL levels were significantly increased in cells stimulated with LPS and PMA compared to unstimulated ones. α-MSH significantly reduced lucigenin CL values and this effect was completely reversed in the presence of indomethacin (10 −8 and 10 −7 M). In conclusion, α-MSH inhibits the production of superoxide radicals by activated rat peritoneal neutrophils and COX contributes to this effect.

Decreased apoptosis of beta 2- integrin-deficient bovine neutrophils.

Stimulant-induced viability of neutrophils, nuclear-fragmentation, increase in intracellular calcium ([Ca2+]i), expression of annexin V on neutrophils and proteolysis of a fluorogenic peptide substrate Ac-DEVD-MCA (acetyl Asp-Glu-Val-Asp alpha-[4-methyl-coumaryl-7-amide]) by neutrophil lysates from five normal calves and three calves with leucocyte adhesion deficiency were determined to evaluate the apoptosis of normal and CD18-deficient neutrophils. Viability was markedly decreased in control neutrophils stimulated with opsonized zymosan (OPZ), compared to CD18-deficient neutrophils at 37 degrees C after incubation periods of 6 and 24 hours. The rate of apoptosis of control neutrophils stimulated with OPZ increased significantly depending on the incubation time, whereas no apparent increase in apoptosis was found in CD18-deficient neutrophils under the same conditions. Aggregated bovine (Agg) IgG-induced apoptosis of control neutrophils was not significantly different from that of CD18-deficient neutrophils. The expression of annexin V on OPZ-stimulated control neutrophils was greater than that of unstimulated ones 6 h after stimulation. No apparent increase in annexin V expression on CD18-deficient neutrophils was found with OPZ stimulation. A delay in apoptosis was demonstrated in CD18-deficient bovine neutrophils and this appeared to be closely associated with lowered signalling via [Ca2+]i, diminished annexin V expression on the cell surface, and decreased caspase 3 activity in lysates.

Maternal serum beta-HCG and alpha-fetoprotein concentrations in singleton pregnancies following assisted reproduction.

The reason for the elevated levels of HCG in assisted reproduction pregnancies remains unknown. Our hypothesis was that this increase is caused by the ovarian superovulation therapy. We compared the beta-HCG and alpha-fetoprotein (AFP) multiples of the median (MoM) in singleton pregnancies after IVF or ICSI with those achieved by frozen embryo transfer (FET) in spontaneous cycles. The HCG and AFP MoMs (plus minus SEMs) of 59 FET pregnancies were compared with 144 IVF (including 48 ICSI) pregnancies. The maternal HCG of pregnancies following ovarian stimulation was 1.31 plus minus 0.08 MoM compared with 1.35 plus minus 0.12 MoM in the unstimulated ones. The values for AFP were 1.06 plus minus 0.05 versus 1.11 plus minus 0.05 respectively. No significant differences could be observed between pregnancies following stimulated IVF/ICSI and unstimulated FET cycles. Our results show that second trimester maternal serum HCG is also elevated in singleton pregnancies following spontaneous FET cycles. The increased maternal serum HCG in IVF pregnancies is thus not related to superovulation therapy. Because of the elevated maternal serum HCG levels, serum screening cannot be performed reliably in pregnancies following assisted reproduction technology. Ultrasonographic detection of the nuchal translucency is unaffected and should be used for this group of women undergoing assisted reproduction.

Insulin up-regulates vascular endothelial growth factor and stabilizes its messengers in endometrial adenocarcinoma cells.

Angiogenesis is crucial for tumor growth and dissemination. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that promotes vascular growth and therefore tumoral growth and metastasis. Overweight, frequently associated with hyperinsulinemia, constitutes the major risk factor for endometrial carcinoma. Thus, elevated insulin levels may partly explain the increased risk of endometrial cancer observed in obese postmenopausal women. The aim of the present work was to test the role of insulin in the control of VEGF expression in endometrial carcinoma cells (HEC-1A). We have shown that insulin induced a biphasic expression of VEGF messenger ribonucleic acid, with an early, but low, induction (4 h of stimulation) and a delayed, but high, induction (24 h). The delayed effect of insulin on VEGF expression involved transcriptional and posttranscriptional regulation, as evidenced by the increased rate of VEGF transcription and the prolonged half-life of VEGF messenger ribonucleic acid. Simultaneously we observed higher levels of VEGF protein in the conditioned medium of stimulated cells compared with unstimulated ones. Therefore, insulin could contribute to the increased risk of endometrial carcinoma due to its ability to induce VEGF expression and thus participate in the maintenance of an angiogenic phenotype.

Effect of carbon-coated polyethylene terephthalate on prostacyclin release by endothelial cells stimulated with arachidonic acid in vitro

The production of 6-kcto prosta-landin Flα (6-keto-PGF1α), stable metabolyte of prostacyclin, by cultured human endothelial cells in contact with carbon- and collagen-coated polyethylene terephthalate (PC), was assessed by enzyme immunoassay. As control material, tissue culture-treated polystyrene was used. The cultures were put in contact with the materials for 48 h and then were stimulated with 0.1 mM arachidonic acid for 3 h. The stimulation induced a highly signilicant increase of 6-keto-PGF 1α in the cultures in contact with the control material. PC induced only insignificant variations in stimulated cultures compared to unstimulated ones. In conclusion, PC determined a decrease in the endothelial cell response to stimulation with arachidonic acid.

Influence of the cold pressor test on the middle cerebral artery circulation

Cold pressor test (CPT) evokes generalized activation of the sympathetic nervous system (SNS). The activity of SNS may be estimated by monitoring the mean blood velocity ( v m) in the middle cerebral artery (MCA) by using a transcranial Doppler monitoring system (TCD). To determine the response of SNS, we studied the v m during CPT. Thirty-four healthy volunteers, 13 female and 21 male (mean age 34±9.5 years, range 18 to 55 years) participated in our study. The experiment consisted of a 5-min baseline period followed by a 3-min immersion of the right hand in ice water. Blood velocity in both MCA's was monitored by bitemporal 2 MHz probes by using a Multi-Dop X4. MAP and heart rate (HR) were measured simultaneously by a Finapres non-invasive blood pressure monitor and a computerized ECG system. End-tidal CO 2 (Et-CO 2) was measured with an infrared capnograph. To determine v m over a chosen time interval the TCD-8 software was utilized. The results showed that during CPT v m, MAP, and HR increased significantly ( P<0.01) for 9.8%, 18.5%, and 3.6%, respectively. Et-CO 2 did not change significantly ( P>0.05). The increase of v m was also significantly higher in the stimulated hemispheres ( P=0.005) regarding to unstimulated ones. The increase of v m during CPT was not gender dependent. To establish the association between variables the models of multivariate regression were used. Multiple regression CPT model was significant ( P<0.01) and fitted data moderately well ( R 2=0.28). MAP and Et-CO 2 were significant in the model ( P<0.01). It seems that the reactivity of the SNS can be estimated by measuring v m with TCD during CPT.

Reduction of Apoptosis of In Vitro Cultured Lymphocytes of HIV-Positive Persons By NG-Hydroxy-Methylated-L-Arginine and 1’-Methyl-Ascorbigen

Some formaldehyde generating chemicals due to reduction of apoptosis in lymphocytes may slow down the progress of immune decline of HIV-infected individuals. N(G)-hydroxy-methylated-L-arginine (MAX) and 1'-methyl-ascorbigen (MeAsc) could enter this way the biochemical pathway of cells and affect the apoptotic process. Separated peripheral blood lymphocytes of five asymptomatic HIV-positive persons were cultured. Unstimulated, IL-2 stimulated and IL-2 stimulated plus 0.1, 1.0, 10.0 microg/ml MAX or MeAsc treated lymphocytes were investigated for apoptosis morphologically (HE) and by flow cytometrical DNA fragmentation method. IL-2 stimulation lowered the apoptotic rate in lymphocytes of HIV-positive persons related to unstimulated ones. MAX and MeAsc reduced the apoptotic activity of stimulated lymphocytes in the least or the middle doses while in the higher dose did not. MAX and MeAsc reduced the apoptotic activity of stimulated lymphocytes originated from HIV-positive patients in vitro. This compounds may have the same effect in vivo and may prolong the symptomless period of HIV-infected patients. The role of methylation and production of formaldehyde in this process is discussed.

Highly sensitive measurement of lipid molecular species from biological samples by fluorimetric detection coupled to high-performance liquid chromatography

As the molecular species composition of glycerophospholipids provides more valuable information than the corresponding fatty acid composition, we have applied a fluorimetric detection (360 and 460 nm for excitation and emission wavelengths, respectively) of anthroyl derivatives of diradylglycerol species to minor phospholipid classes and subclasses from biological samples. Diacylglycerol species were obtained by phospholipase C treatment of phosphatidylcholine subclasses and phosphatidic acid extracted from rat thymocytes. Subpicomole measurements of molecular species from the minor subclass alkenylacylglycerophosphocholine could be achieved (e.g. 0.4 pmol of the 18:1/20:5 species). Such a sensitivity allowed study of the molecular species composition of another minor phospholipid, phosphatidic acid, and to evaluation of its alteration in mitogen-stimulated thymocytes as compared to unstimulated ones. Finally, we report that such a measurement is also applicable to other minor bioactive lipids with a hydroxyl group available, namely hydroxyeicosatetraenoates (HETEs), with a similar gain of sensitivity over conventional UV detection. Overall, these measurements, especially those of phospholipid molecular species, are sensitive, reliable and meaningful for precursor–product relationship between phospholipids.

Peptide antigen or superantigen-induced down-regulation of TCRs involves both stimulated and unstimulated receptors.

T cell activation by peptide/MHC complexes, superantigens, or mAbs induces the down-regulation of cell surface TCRs. We addressed the question of whether TCR down-modulation affects only TCRs that had directly interacted with their ligand or whether down-modulation could also affect TCRs that had not interacted with their ligand. To this end, we generated T cells coexpressing equal levels of two different TCRs by transfecting the appropriate cDNAs into cells of the human T cell line, Jurkat. Each set of TCRs can be distinguished by means of anti-Vbeta mAbs and can be stimulated separately with peptide Ag, bacterial superantigens, or mAbs. We found that activation of these cells with each of these stimuli down-modulated not only directly stimulated TCR complexes but also unstimulated ones. Comodulation of stimulated and unstimulated receptors may reflect functional interactions between surface TCRs that could take place during Ag or superantigen recognition by T cells without the need for ligand cross-linking. Consistent with this idea, both stimulated and unstimulated receptors colocalized in patches on the cell surface after activation.

Up-regulation of thrombomodulin is inducible on an endothelialized polyester.

The concept of endothelial cell seeding of vascular prostheses is designed as a method for improving long-term patency substitutes because endothelium is considered as the haemocompatible surface of reference. The assessment of the functionality of cells lining a biomaterial is thus of crucial importance. We have reported encouraging results concerning the ability of a polyester coated with albumin and chitosan (M 11) to be lined by a confluent monolayer of cultured human endothelial cells (EC). The aim of the present work was to study the expression of thrombomodulin (membrane glycoprotein responsible for anticoagulant activity) in EC lying on M.11 by anticoagulant activity and mRNA level with and without stimulation. Results obtained in basal conditions showed that EC on M.11 have a comparable expression of TM mRNA when compared to control (EC on tissue culture plates) despite a lower TM surface activity for EC on the biomaterial. In terms of ratio (stimulated cells to unstimulated ones) the response in activity for EC on M.11 is comparable to that of the control. These results indicate that cells lying on M.11 are able to respond to physiological-like stimuli, despite a tendency for these cells to express a procoagulant phenotype when compared with control EC.

Enhancement of B Cell Function as Antigen-Presenting Cell during Interplay with Th Cells

We have previously reported that C57BL/6 mouse B cells present Ag to an I-A b-restricted and OVA-specific T cell clone, 34-7F, to secrete IL-2 but not to proliferate, whereas spleen adherent cells as APC induce a proliferative response of 34-7F cells to OVA. In this T cell response, it was examined whether B cells were stimulated during presenting Ag and acquired the ability as APC to induce proliferative Ag-response of 34-7F cells. For this aim, B cells prepared from unstimulated mouse spleen cells were cultured with 34-7F cells in the presence of OVA and then compared with unstimulated B cells for APC function in the OVA-response of 34-7F cells. Unstimulated B cells proliferated when cultured for 2 days with irradiated 34-7F cells and OVA, and the proliferation was inhibited by an anti-I-A b mAb. B cells which had been stimulated for 2 days with 34-7F cells and OVA induced an increase in phosphatidyl inositol metabolism in 34-7F cells in the presence of Ag, whereas unstimulated B cells failed to do so. The increase was dependent on the dose of OVA and on the number of the stimulated B cells. Ag presentation by the stimulated B cells (but not by unstimulated ones), also induced IL-2R expression on 34-7F cells, which resulted in IL-2-dependent proliferation. The IL-2R expression does not depend on the possible increase in IL-2 production by the T cells, inasmuch as the activated B cells induced lower expression of IL-2 mRNA or lower secretion of IL-2 by 34-7F cells than that induced by unstimulated B cells. These results suggest that B cells receive signal(s) from 34-7F cells to enhance APC function during Ag-presentation to the T cells which do not proliferate in the response to Ag on unstimulated B cells, and that the stimulated B cells induce the proliferative response of 34-7F cells to OVA.