PKC cellular distribution in TPA activated human peripheral blood mononuclear cells, treated with an anti-HLA class I monoclonal antibody

Cytosolic and Particulate Protein Kinase C has been studied in Peripheral Blood Mononuclear Cells activated with 12-O-Tetradecanoyl phorbol 13-acetate and treated with the anti-HLA Class I Monoclonal Antibody 01.65. No effects on the cellular distribution of PKC activity nor to the proliferative response has been found. In phytohemagglutinin stimulated PBMC cultures treated with MoAb 01.65 total PKC activity depletion and 3H-Thymidine incorporation inhibition has been found. In PBMC cultures activated with both PHA and TPA, the proliferative response was similar to cultures activated with PHA alone, while the PKC cellular distribution was similar to the one detected in TPA stimulated cultures. Addition of the MoAb 01.65 was ineffective on both PKC activity and 3H-Thymidine incorporation. These data indicate that anti-HLA Class I MoAb induced 3H-Thymidine incorporation inhibition may be related to low levels of PKC activity.     

Induction of cytotoxicity in human peripheral blood mononuclear cells by monoclonal antibody OKT3

It has been previously reported from this laboratory that incubation of PBMC with OKT3 generates potent cytotoxic lymphocytes that can be targeted by using antibody heteroconjugates consisting of anti-target cell antibody and OKT3. In the present study these conjugates were used to explore the kinetics of induction of cytotoxicity in PBMC and the subpopulations of lymphocytes involved. It was found that in addition to conjugate-dependent cytotoxicity, a considerable amount of conjugate-independent cytotoxicity was generated during OKT3 stimulation. Although the conjugate-dependent activity resided in the CD8+ population, the conjugate-independent cytotoxicity was found to be a function of CD4-/CD8- natural killer-like cells. Being largely CD3-, those cells were most likely activated by lymphokines produced by OKT3-stimulated CD3+ cells. They were capable of killing not only tumor cells but also autologous lymphocytes. The CD4+ cells of some donors were found to exhibit low but clearly demonstrable cytotoxicity. Induction of cytotoxicity was characterized as an early event in T cell activation, correlating with the kinetics of RNA synthesis. Cytotoxicity, interleukin 2 receptor expression, and DNA synthesis declined after 3 days of activation with OKT3, indicating the existence of as yet undefined regulatory mechanisms.     

Depletion of protein kinase C induced by an anti HLA class I monoclonal antibody in phytohemagglutinin activated human T cells

Anti HLA Class I Monoclonal Antibody depletes Protein Kinase C (PKC) to 20% of control value in PHA activated human T cells. The effect is reversible: in 24 hours the enzymatic activity returns to 58% of control value. Removal of antibody from the culture medium increases the rate of recovery. Implications of this finding for the modulation by HLA Class I antigens of the proliferative response of T cells to lectins are discussed.     

Competitive effect of anti-HLA class I monoclonal antibody (01.65) and N-N-staurosporine on prolipherative response of PHA activated T-lymphocytes

N-N-Staurosporine (STAR) inhibits in a dose dependent manner Tritiated-Thymidine (3H-TdR) incorporation in phytohemagglutinin (PHA) activated Peripheral Blood Mononuclear Cells (PBMC) treated with anti-HLA class I monoclonal antibody (MAb) 01.65 and its effect results competitive with MAb 01.65. Cytosolic and particulate Protein Kinase C (PKC) have been studied. Only when PKC particulate activity is no more detectable, the effect of STAR on 3H-TdR incorporation is evident.     

Major histocompatibility complex class I restricted T-cell autoreactivity in human peripheral blood mononuclear cells

During selection in the thymus or any subsequent response, T-cells recognize peptides bound to major histocompatibility complex (MHC) molecules. Peptides produced by lysosomes or by proteasome/immunoproteasome stimulate CD4+ or CD8+ T-cell, respectively. Inflammation alters components of both antigen-processing pathways resulting in the production of different peptides. The role of such changes in self/non-self discrimination was examined in autologous mixed peripheral blood mononuclear cell cultures. Stimulator cells were incubated in the presence or absence of INF-gamma, with or without lysosome inhibitors (ammonium chloride/chloroquine), cathepsin inhibitor (E-64), or proteasome/immunoproteasome inhibitor (epoxomicin). Responder cells were added and zeta-chain phosphorylated forms were used as read out. INF-gamma did not affect zeta-chain phosphorylated forms, which means that the expected INF-gamma induced alterations in antigen processing machinery do not influence self/non-self discrimination. Surprisingly, the completely phosphorylated 23-kDa zeta-chain was always present except in the case of epoxomicin, indicating the presence of MHC class I restricted autoreactive CD8+ T-cells but not of MHC class II restricted autoreactive CD4+ T-cells, possibly due to more efficient negative selection in the thymus of the latter. Autoimmunity is prevented due to absence of help by CD4+ T-cells. This conclusion was confirmed by the lack of differences in IL-2 levels in cell culture supernatants, as well as, by the absence of differences in cell proliferation under the various conditions described above.     

Non-specifically activated human peripheral blood mononuclear cells are cytotoxic for human keratinocytes in vitro

Cultured human keratinocytes were lysed by activated PBMC in a 4-h 51Cr release assay. PBMC were activated by incubation with 50 U/ml of rIL-2 for 4 days. The cytotoxic precursors were found to be NKH1+ and included both CD2+ and CD2- phenotypes. This cytotoxicity was not genetically restricted, as cells killed both allogeneic and autologous keratinocytes without priming. Cytotoxicity was blocked by pre-incubation of effector cells with mAb against LFA-1 alpha-(TS1/22) and beta-chains (TS1/18), but not by antibodies directed against CD4, CD8, or leukocyte common Ag (T200) suggesting that LFA-1 is an important interactive molecule in this cytotoxicity. IFN-gamma is reported to upregulate ICAM-1, the ligand for LFA-1. Pre-treatment of target keratinocytes with IFN-gamma was also found to greatly increase the sensitivity of keratinocytes to lysis. This increased sensitivity to lysis was blocked by anti-LFA-1 and anti-ICAM-1, but not by anti-DR (L243), and thus was not the result of increased DR expression. Such treated targets were lysed at low levels (15 to 18%) by an Ag-specific CD8+ cytotoxic clone as well as a T cell line derived from a skin lesion of allergic contact dermatitis. In contrast, control keratinocytes were only sensitive to IL-2-activated PBMC as described above. The above findings may be relevant to a variety of conditions in which epidermal damage is associated with lymphocytic infiltrate. These conditions include graft-vs-host disease, erythema multiforme, and lupus erythematosus. DR+ keratinocytes, which may be a marker for IFN-gamma are also found in the above conditions. It is suggested that epidermal pathology may be mediated by non-specific cytotoxicity induced in the course of an immune response.     

Endothelial cells co-stimulate peripheral blood mononuclear cell responses to monoclonal antibody TGN1412 in culture

The failure of preclinical testing to predict the severity of the cytokine storm experienced by the recipients of the superagonistic anti-CD28 monoclonal antibody (mAb) TGN1412 during its Phase 1 clinical trial prompted the development of new in vitro experimental approaches for mimicking in vivo cytokine release and lymphoproliferation. Peripheral blood mononuclear cells (PBMC) presented to TGN1412 immobilised on plastic has previously been shown to stimulate a pro-inflammatory cytokine response. The aim of the present study was to investigate a 'co-culture' model for the detection of TGN1412-like immunomodulatory activity in which TGN1412 was presented to PBMC in the presence of monolayers of endothelium-derived cells and other cell types, followed by measurement of cytokine levels in the culture supernatants and proliferation of PBMC. Culturing PBMC with TGN1412 over primary human umbilical vein endothelial cells (HUVEC) and HUVEC-derived cell lines retaining classic endothelial markers, but not cell lines of non-endothelial origin, mediated the specific release of IL-6, IL-8 and TNFα, and proliferation of PBMC. Low levels of IL-2 and IFNγ were also detected in supernatants with most donors of PBMC. An anti-CD28 mAb agonist, i.e., not a superagonist like TGN1412, did not stimulate cytokine release or proliferation of PBMC in co-cultures. In conclusion, co-culture experiments for TGN1412-specific cytokine release required cells of endothelial origin. However, the profile of released cytokines in co-cultures did not mirror that in the clinical trial participants or the responses from PBMC exposed to TGN1412 immobilised on plastic, suggesting that TGN1412 stimulation of PBMC can occur through more than one mechanism.     

Ovine interleukin 12 has biological activity on ovine and human activated peripheral blood mononuclear cells

Interleukin 12 (IL-12) is a heterodimeric cytokine composed of two subunits that form the biologically active p70 molecule, and is a potent inducer of the pro-inflammatory cytokine IFN-gamma. In this study the coding sequence for ovine interleukin 12 p35 and p40 subunits was derived by RT-PCR cloning. Ovine p35 and p40 cDNA sequences show a high level of similarity at the nucleic acid and protein levels when compared to corresponding bovine and human sequences. In particular, cysteine residues and N-linked glycosylation sites are conserved between species. Secretion of the IL-12 heterodimer from CHO cells co-transfected with ovine p35 and p40 cDNA was shown by immunoprecipitation of a 60 and 66 kDa protein from transfectant supernatant. In addition, the supernatant from co-transfected cells augmented the proliferation of Con A-activated ovine peripheral blood mononuclear cells (PBMNC). Cross-species activity was shown by the enhancement of proliferation of human phytohaemagglutinin (PHA)-activated PBMNC. Supernatants from co-transfectants of hu p35/ov p40 and ov p35/hu p40, to generate chimeric heterodimers, also demonstrated stimulatory activity. Human and chimeric IL-12-induced proliferation of activated PBMNC was inhibited using an anti-human IL-12 polyclonal antibody, however this antibody showed minimal inhibition of ovine IL-12. This study suggests that ovine IL-12 has biological properties similar to its human counterpart.     

Anti-peptide antibody production elicited by in vitro immunization of human peripheral blood mononuclear cells

Human monoclonal antibodies have great potential for use in the treatment of various diseases. We have established an in vitro immunization protocol for inducing antigen-specific antibody production from human peripheral blood mononuclear cells (PBMCs). In the in vitro immunization protocol, PBMCs are pretreated with L-leucyl-L-leucine methyl ester (LLME) to remove suppressive cells, and are sensitized and cultured with a soluble antigen in the presence of IL-2, IL-4 and muramyl dipeptide for 8 d, and then an antigen-specific antibody is produced. In this study, we examined the novel possibility of an in vitro immunization protocol, specifically, whether LLME-treated PBMCs can be sensitized with a peptide antigen to produce an anti-peptide antibody. The results indicate that antigen-specific immune responses were elicited by a peptide antigen derived from rice allergen, a cholera toxin B subunit, and TNF-alpha as a sensitizing antigen in in vitro immunization. These results suggest that the in vitro immunization protocol is applicable in the generation of an anti-peptide antibody against various antigens, including food allergens, foreign antigens, and self-antigens.     

Altered proliferative kinetics in PHA-activated human T-lymphocytes treated with the anti-HLA class I monoclonal antibody 01.65

Abstract. Anti-HLA class I monoclonal antibody (mAb) 01.65 inhibited phyto-haemagglutinin (PHA)-induced human lymphocyte proliferation. The inhibitory effect was inversely correlated to the strength of the proliferative response. It was increased when lymphocytes were stimulated with suboptimal doses of PHA but it disappeared with supraoptimal doses. Proliferation inhibition was achieved by prolonging the cell cycle time and by slowing down its recruitment rate. The former effect was not restricted to the G₁-phase but also included the S phase. These results support the idea that HLA class I molecules are important in the PHA-induced proliferation of human T-lymphocytes.     

Spontaneous DNA repair in human peripheral blood mononuclear cells

DNA molecules are constantly damaged during mitosis and by oxygen-free radicals produced by either cellular metabolism or by external factors. Populations at risk include patients with cancer-prone disease, patients under enhanced oxidative stress, and those treated with immunosuppressive/cytotoxic therapy. The DNA repair process is crucial in maintaining the genomal DNA integrity. The aim of this study was to evaluate spontaneous DNA repair capacity of peripheral blood mononuclear cells (PBMC) from normal blood donors. PBMC DNA repair ability represents DNA repair by other tissues as well. It is shown in the present study that in vitro incorporation of [3H]thymidine in non-stimulated PBMC expresses the ability of the cells to repair DNA damage. This method was validated by double-stranded DNA measurements. Both catalase and Fe2+ increased DNA repair, the former by preventing re-breakage of newly repaired DNA and the latter by introducing additional DNA damage, which enhanced DNA repair. Better understanding of DNA repair processes will enable to minimize DNA damage induced by oxidative stress.     

Helper activity for antibody synthesis encoded by mRNA extracted from human peripheral blood mononuclear cells

Translation products in Xenopus laevis of mRNA from human peripheral blood mononuclear cells were tested for their capacity to replace T cells in the anti-SRBC response of nude spleen cells. When the starting material came from PHA or FCS-stimulated lymphocytes, the translated lymphokines, displaying such a B cell helper activity were found to be encoded by mRNA sedimenting at 6-7S and 13S on a sucrose density gradient. 6-7S mRNA from control, non-stimulated lymphocytes was also able to code for B cell helper activity. Thorough T-cell depletion of mouse responder cell populations left unchanged the activity of 6-7S mRNA products while preventing that of 13S products. The latter were found to contain IL-2, which suggests that their action on B cells was indirect, mediated by T-cell stimulation.     

Cholesterol modulates P-glycoprotein activity in human peripheral blood mononuclear cells

P-glycoprotein (P-gp) is expressed in a wide range of cell types including peripheral blood mononuclear cells (PBMCs) where it may restrict intracellular accumulation of substrates like antineoplastic agents, HIV protease inhibitors, or rhodamine123. P-gp is known to be located in membrane microdomains, whose structure and function are susceptible to cholesterol alterations. This study evaluated the effect of cholesterol alteration in human PBMCs on P-gp activity. Whereas cholesterol depletion had no effect, cholesterol repletion of depleted cells significantly decreased intracellular rhodamine123 concentrations in lymphocytes to 32.2%+/-2.7 (p<0.001) and to 41.9%+/-3.5 (p<0.001) in monocytes. After cholesterol saturation of native cells intracellular rhodamine123 fluorescence decreased to 12.4%+/-1.6 (p<0.001) in lymphocytes and 12.9%+/-3.5 (p<0.001) in monocytes. These data demonstrate that elevated cellular cholesterol levels can markedly increase P-gp activity in human PBMCs.     

Lack of a role of monocytes in the inhibition by monoclonal antibodies to monomorphic and polymorphic determinants of HLA class I antigens of PHA-P-induced peripheral blood mononuclear cell proliferation

This study aimed at characterizing the mechanism(s) underlying the regulatory role of distinct determinants of HLA Class I antigens in PHA-P-induced T cell proliferation and the involvement of monocytes in this phenomenon. The anti-HLA-A2,A28 monoclonal antibodies (MoAb) CR11-351, the MoAb Q6/64 to a determinant restricted to the gene products of the I antigens HLA-B locus, and the MoAb CR10-215 and W6/32 to distinct monomorphic determinants of HLA Class I antigens were found to inhibit PHA-P-induced peripheral blood mononuclear cell (PBMC) proliferation in a dose-dependent fashion. The inhibition is specific and reflects neither inhibition of PHA-P binding to cells nor a toxic effect of the anti-HLA Class I MoAb. The latter differed in the concentration required to induce inhibition, in the influence of the concentration of PHA-P used as mitogen, in the differential effect on the donors used as a source of PBMC, and/or in the requirement of the Fc portion to induce inhibition. At variance with the information in the literature, the inhibitory effect of anti-HLA Class I MoAb on PHA-P-induced PBMC proliferation neither reflected their interaction with accessory cells nor was mediated by suppressor factors released by monocytes stimulated with PHA-P in the presence of anti-HLA Class I MoAb. Therefore, the regulatory role of HLA Class I antigens in T cell proliferation is not likely to be mediated by monocytes and/or factors released from them, but may reflect an involvement of these molecules in T cell activation pathways.     

Development of an 8-color antibody panel for functional phenotyping of human CD8+ cytotoxic T cells from peripheral blood mononuclear cells

The study of CD8 positive cells in peripheral blood has become an essential part of research in the field of cancer immunotherapies, vaccine development, inflammation, autoimmune disease, etc. In this study, an 8-color flow cytometry panel, containing lineage and functional markers, was developed for the identification of CD8+ cytotoxic T cells in previously cryopreserved peripheral blood mononuclear cells from healthy human donors. By studying functional markers in naïve and CD3/CD28 activated T cells we demonstrate that the panel is capable of detecting protein markers corresponding to different T cell activation statuses. Data generated by flow cytometry were corroborated by different antibody based assay technologies to detect soluble cytokines. Our findings suggest that there is an inter donor variability in both baseline and activation responses. We have also successfully developed an antibody panel for flow cytometry that could be used to study cytotoxic function of CD8 T cells in clinical immunology research areas.     

Analysis of mRNA for class I HLA on human gametogenic cells

We have studied mRNA expression for Class I HLA (human leukocyte antigen) on male germ cells by amplification of gene fragments in PCR techique and by Northern hybridization. RNA was extracted from fractionated gametogenic cells (isolated from testis) and reversely transcribed. Then, cDNA was amplified for specific HLA sequence (1151 bp) representing whole-length coding sequence (HLA, -A, -B, -C). The specificity of this product was confirmed in “nested” PCR of 400 bp gene fragment coding for alpha 2 domain, alpha 3 domain, and the transmembrane portion of Class I HLA. The results indicate minimal expression of classical Class I HLA on gametogenic cells. Northern hybridization with 669 bp cDNA fragment (spanning for alpha 3 domain, transmembrane, cytoplasmic, and 3′ untraslated region) resulted in a low intensity signal from gametogenic cell fractions and confirmed our findings obtained by PCR. The minimal expression of classical HLA antigens may create a neutral cover for the male reproductive system, thereby preventing an immunological response during germ cell differentiation. © 1994 Wiley-Liss, Inc.     

C-fos, c-myc and IL-2R mRNA expression in PHA activated T lymphocytes treated with a monoclonal anti-HLA class I antibody (MAb 01.65)

Monoclonal anti HLA class I antibodies inhibit the proliferative response of PHA-stimulated T lymphocytes. We studied the effects of MAb 01.65 anti-HLA class I on c-fos, c-myc and IL-2R mRNA expression. We found that MAb treatment does not modify either c-fos mRNA levels observed after 10 minutes to 3 hrs or the early c-myc mRNA expression revealed after 1 to 6 hrs, but decreases the intensity of autoradiographic signals of late c-myc and IL-2R mRNA expression. Since we had previously ascertained that MAb 01.65 treatment induces a decrease in PKC enzymatic activity after few minutes, the correlation of that result with the data presented in this paper will be discussed.