Anti HLA class I monoclonal antibody effect on PKC kinetics in PHA activated human peripheral blood mononuclear and E+ cells

Cytoplasmic protein kinase C (PKC) has been studied in phytohemagglutinin (PHA) activated peripheral blood mononuclear cells (PBMC) and macrophage depleted E+ cell culture. Within 10' after contemporanous addition of PHA and anti HLA class I monoclonal antibody 01.65 (MoAb) PKC is depleted in both cell types. Enzyme activity recovers in the following hours however at 72 hours is at control values in E+ cultures while in PBMC cultures it is still depleted at 68% of the control. Anti HLA class I MoAb induced tritiated lymidine (3H-TdR) incorporation inhibition appears to be related to low levels of PKC activity.     

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.     

Molecular analysis of cell cycle-related gene expression in anti-HLA class I monoclonal antibody (01.65) treated PHA-activated human T-lymphocytes.

HLA class I antigens seem to be involved in the proliferative response of PHA-activated human T-lymphocytes. We have previously reported that the treatment of PHA-activated peripheral blood mononuclear cells (PBMC) with an anti-HLA class I monoclonal antibody, 01.65, (i) inhibits the tritiated thymidine incorporation, (ii) inactivates cytosolic protein kinase C (PKC) and (iii) causes an increase in the duration of the cell cycle. Northern Blot kinetic analysis of c-fos, c-myc, cdc2, IL-2R, c-myb, ODC, TK and H3, from 10 minutes to 120 hours, was performed in MAb 01.65 treated cultures. We found that the expression of four genes (c-myc, IL-2R, cdc2 and TK) was depressed 24 hours after PHA stimulation.     

An anti-HLA class I monoclonal antibody alters the progression in the cell cycle of phytohemagglutinin-activated human T lymphocytes

The monomorphic anti-HLA Class I monoclonal antibody 01.65 inhibits the incorporation of tritiated thymidine ([3H]TdR) in Phytohemagglutinin (PHA)-activated human T lymphocytes. Our data indicate that 01.65 affects the average duration of the cell cycle by increasing the length of the early S subphase. As a consequence of the increase in the doubling time of the cell population, the absolute number of cells at harvesting time was reduced in 01.65-treated cultures compared to that of untreated cultures. The lengthening of the S-phase and the decrease in the cell number can together quantitatively account for the reduction of [3H]TdR incorporation observed in 01.65-treated cultures.     

Anchored anti-HLA class I monoclonal antibody fails to induce inhibition of PHA-activated lymphocytes proliferation.

It is known that anti-HLA Class I antibodies inhibit the proliferative response of PHA-activated T-lymphocytes. We found that plastic- or sepharose-linked anti-HLA Class I monoclonal antibody 01.65 does not inhibit either [3H]Thymidine incorporation or recruitment in the cell cycle, nor does it reduce the expression of c-myc mRNA and the membrane expression of Interleukin-2 Receptor and Transferrin Receptor. Furthermore, particulate Protein Kinase C is not affected by anchored anti-HLA Class I monoclonal antibody 01.65. We suggest that anti-HLA Class I monoclonal antibody may act through crosslinking or internalization of HLA Class I antigens.     

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.     

Regulatory role of a monomorphic determinant of HLA Class I antigens in T cell proliferation

The role of HLA Class I antigens in T cell proliferation was investigated by using the anti-HLA Class I monoclonal antibodies (MoAb) CR10-215, CR10-325, and CR11-115. MoAb CR10-215 and CR11-115 recognize the same (or spatially close) monomorphic determinant, which is distinct and spatially distant from that reacting with MoAb CR10-325. Addition of MoAb CR10-215 and CR11-115 to cultures of peripheral blood mononuclear cells stimulated with MoAb OKT3, MoAb Pan T2, PHA, or PPD inhibited cell proliferation. The blocking is specific in that the anti-HLA Class I MoAb CR10-325 and the Pan T MoAb Pan T1 had no effect on the proliferation. The inhibitory activity of MoAb CR10-215 and CR11-115 does not reflect i) toxic effects, ii) induction of suppressor cells and factors, iii) blocking of the binding of mitogens to lymphocytes, iv) inhibition of the production of interleukin 1 (IL 1) and interleukin 2 (IL 2), or v) function of IL 2 receptor. Anti-HLA Class I MoAb were able to inhibit the proliferation of purified, Tac-, T cells. The inhibited cells did not express Tac antigen, as assayed by direct immunofluorescence, with MoAb anti-Tac, but released a normal amount of IL 2 in culture medium. These results indicate that monomorphic determinants of the HLA Class I complex are involved in the regulation of T cell proliferation. The effect appears to occur at the level of IL 2 receptor expression.     

Role of HLA class I and class II antigens in activation and differentiation of B cells

The monoclonal antibodies (MoAb) CR10-214, CR11-115, and Q1/28 to distinct monomorphic determinants of HLA class I antigens, the MoAb CL413 and PTF29.12 recognizing monomorphic determinants of HLA-DR antigens, the anti-HLA-DQw1 MoAb KS11, the anti-HLA-DPw1 MoAb B7/21, and the anti-HLA-DR,DP MoAb CR11-462 were tested for their ability to modulate human B-lymphocyte proliferation and maturation to IgM-forming cells. Purified tonsillar B cells were stimulated with Staphylococcus aureus bacteria of the Cowan first strain (SAC) or anti-human mu-chain xenoantibodies, as well as in growth factor- or T-cell-dependent activation cultures. The B-cell proliferative responses induced by SAC or by mitogenic concentrations of anti-mu-chain xenoantibodies were inhibited by some of the anti-HLA class I and anti-HLA class II monoclonal antibodies tested. The same antibodies were effective inhibitors of the proliferation of B cells stimulated with interferon-gamma (IFN-gamma) or interleukin-2 (IL-2) and with submitogenic concentrations of anti-mu-chain xenoantibodies. The proliferation induced by IL-2 of SAC-preactivated B cells was inhibited by some of the anti-HLA class II monoclonal antibodies, but not by the anti-HLA class I monoclonal antibodies tested. This inhibition appeared to reflect at least in part a direct effect on later events of the B-cell activation cascade, since some anti-HLA class II monoclonal antibodies still exerted considerable inhibitory activity when added together with IL-2 to SAC-preactivated B cells after the third day of culture. Anti HLA-DR, DQ, and DP monoclonal antibodies consistently inhibited the IgM production induced in B cells by T cells alone, T cells plus pokeweed mitogen (PWM), SAC plus IL-2, or IL-2 alone. In contrast, two of the three anti-HLA class I monoclonal antibodies tested inhibited the IgM production in cultures stimulated with SAC plus IL-2 and one the IgM production induced by IL-2 alone, but none of them had inhibitory effects on T-cell dependent IgM production. The results reported herein indicate that HLA class II molecules directly participate in different phases of the B-cell activation cascade. In addition, our data also suggest that HLA class I molecules can be involved in the events leading to B-cell proliferation and differentiation into immunoglobulin-secreting cells.     

Antibody-induced association between discrete regions of HLA class I and II antigens

The anti-HLA-DR + DP monoclonal antibody (MoAb) CR11-462 was unexpectedly found to cross-inhibit the binding to B lymphoid cells of the anti-HLA Class I MoAb CR10-215 and CR11-115. The latter two antibodies recognized the same or spatially close antigenic determinant. The cross-blocking of anti-HLA Class I MoAb CR10-215 and CR11-115 by MoAb CR11-462 reflects neither its contamination by anti-HLA Class I antibodies nor its cross-reactivity with HLA Class I antigens. On the other hand, the cross-blocking appears to reflect redistribution of HLA Class II antigens by the MoAb CR11-462, since the MoAb CR10-215 and CR11-115 are not susceptible to blocking when lymphoid cells are treated with 0.025% glutaraldehyde or are coated with Fab' fragments of the MoAb CR11-462. Furthermore, immunoprecipitates from B lymphoid cells preincubated with the MoAb CR11-462 before solubilization contain HLA Class I antigens. Therefore, these results have shown for the first time an antibody-induced association between discrete regions of HLA Class I and Class II antigens on the membrane of B lymphoid cells.     

Regulatory role of monomorphic and polymorphic determinants of HLA class I antigens in the proliferation of T cells stimulated by autologous and allogeneic B and T lymphoid cells

Monoclonal antibodies (mAb's) to monomorphic and polymorphic determinants of HLA Class I antigens were shown to inhibit proliferation of T cells stimulated with autologous and allogeneic B and T lymphocytes. Inhibition of proliferative responses was lower when T cells were used as stimulators than when B cells were used. The inhibitory activity was similar for mAb's to monomorphic and polymorphic determinants of HLA Class I antigens, suggesting that the density of antigen-antibody complexes on the cell membrane does not play a major role in the phenomenon. The anti-HLA Class I mAb's exerted their inhibitory effect at the level of both the responding and the stimulating cells. Addition of exogenous interleukin 2 to the mixed cultures did not affect the mAb-mediated inhibition.     

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.     

Stimulation and inhibition of human T cell subsets by wheat germ agglutinin

Wheat germ agglutinin (WGA), a tetravalent lectin, has both stimulatory and inhibitory effects on human T lymphocytes. It has been suggested that these actions are related and that WGA selectively stimulates a suppressive subset of T cells. We studied the ability of WGA to stimulate and inhibit subpopulations of human peripheral blood mononuclear cells (PBMC) known to have helper or suppressor activity. Fresh human PBMC were depleted of either T4+ or T8+ cells by using antibody-mediated complement lysis. The resultant cell populations were stimulated with WGA, and the proliferative response was assessed by [3H]thymidine incorporation, IL 2 receptor expression, the ability to elaborate IL 2 in culture supernatants, and the susceptibility to inhibition by the monoclonal antibody anti-Tac. Similar experiments with cells from a WGA-responsive continuous T cell culture were also performed. WGA inhibited phytohemagglutinin (PHA)-induced proliferation of PBMC depleted of either T4+ or T8+ cells. WGA also inhibited PBMC that had been depleted of adherent cells and Ia+ cells and then induced to proliferate with a combination of TPA and PHA. Our findings indicate that WGA induces IL 2-dependent proliferation in a small proportion of both T4+ and T8+ lymphocytes. We also provide evidence that the inhibitory activity of WGA is not mediated by a T4+, T8+, or Ia+ cell, suggesting that WGA acts directly on the proliferating cell rather than selectively stimulating a suppressive subpopulation.     

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.     

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.     

Syngeneic anti-idiotypic antisera to murine anti-HLA class II monoclonal antibodies

BALB/c mice have been immunized with six anti-HLA Class II monoclonal antibodies (MoAb); the latter included MoAb CR11-462, Q5/6, and Q5/13 to monomorphic determinants, the anti-HLA-DR1,4,w6,w8,w9 MoAb AC1.59, the anti-HLA-DRw9 MoAb AB7ae9, and the anti-HLA-DQw3 MoAb AC6G. The six monoclonal antibodies markedly differ in their ability to induce anti-idiotypic antibodies, because the latter were not detected in the sera from the mice immunized with the MoAb AB7ae9 and with the MoAb AC6G. The MoAb AC1.59 and CR11-462 elicited antibodies to private idiotypes, and the MoAb Q5/6 and Q5/13 elicited antibodies to private and public idiotypes. The titer of the latter in the anti-MoAb Q5/6 antiserum appears to be markedly lower than that of the former ones; no marked difference was detected in the titer of the two types of antibodies in the anti-MoAb Q5/13 antiserum. Blocking experiments with a panel of monoclonal antibodies showed that the MoAb Q5/13 shares idiotypes with the anti-HLA Class I MoAb Q5/6, 127, and 441 and with the anti-HLA Class I MoAb CR11-351, Q1/28, Q6/64, and 6/31, but does not share idiotypes with any of the nine anti-human melanoma-associated antigen MoAb tested. The spectrotypes of the anti-MoAb CR11-462 and anti-MoAb Q5/13 antisera comprise two major components in the pH 6.2 to 6.7 range, that of the anti-MoAb Q5/6 antiserum comprises two major components in the pH 6.5 to 6.8 range, and that of the anti-MoAb AC1.59 antiserum comprises a number of components in the pH 5.6 to 7.2 range.     

Effect of Agrobacterium tumefaciens (Smith and Town). Conn. on the functional properties of human blood lymphocytes

Agrobacterium tumefaciens has been shown to affect the proliferation of intact lymphocytes as well as lymphocytes stimulated by PHA action. Inoculation of bacteria into the cultures of intact lymphocytes and incubation during 72 h resulted in increased incorporation of [3H]-thymidine into the DNA of cultivated cells. The bacteria are capable of inhibition of lymphocytes proliferation stimulated by PHA.     

Phospholipase D in cultured rat vascular smooth muscle cells and its activation by phorbol ester

We determined the phospholipase D (PLD) activity in rat vascular smooth muscle cells by the formation of phosphatidylethanol in cells prelabeled with [3H] myristic acid. The enzyme was markedly activated by a phorbol ester (TPA). Down regulation of protein kinase C (PKC) resulted in almost complete inhibition indicating PKC-dependent mechanism of its activation. Depletion of calcium by EGTA and TMB-8 caused 53% inhibition. Chelator-stable association of PKC to membrane by TPA was observed in the absence of extracellular Ca2+. The mitogenic peptide PDGF also caused a marked stimulation of PLD. These results indicate that PLD in vascular smooth muscle cells is stimulated by TPA through the activation of PKC both by calcium-dependent and independent mechanisms.     

Enhancing effect of anti-HLA class I monoclonal antibodies on T cell proliferation induced via CD2 molecule

The mAb 131 to a determinant preferentially expressed on the gene products of the HLA-A locus, the mAb Q6/64 and 4E to determinants preferentially expressed on the gene products of the HLA-B locus, the anti-HLA-A2,A28 mAb CR11-351, HO-2, HO-3, HO-4, and KS1, and the anti-HLA-B7 cross-reacting group mAb KS4 enhanced proliferation of T cells in most, if not all, the PBMC preparations stimulated with the anti-CD2 mAb 9-1 + 9.6. The mAb CR10-215, W6/32, and 6/31 to distinct monomorphic determinants of HLA class I antigens enhanced CD2-induced T cell proliferation in at most 30% of the PBMC preparations tested. The anti human beta 2-microglobulin (beta 2-mu) mAb NAMB-1 displayed no detectable effect on the proliferation of T cells stimulated with the mAb 9-1 + 9.6. The enhancing effect of anti-HLA class I mAb is specific, is dose dependent, is not abrogated by the addition of exogenous IL-1 and IL-2 to the cultures, and reflects the interaction of anti-HLA class I mAb with T cells. Enhancement of CD2 mediated proliferation of T cells is not a unique property of anti-HLA class I mAb, since the anti-HLA class II mAb Q5/6 and Q5/13 also had a similar effect. Analysis of the kinetics of the enhancing effect of anti-HLA class I mAb suggests that they modulate an early event of T cell activation and may affect the interaction of T cells with mAb 9-1. Phenotyping of T lymphocytes activated by mAb 9-1 + 9.6 in the presence of anti-HLA class I mAb suggests that the enhancing effect of anti-HLA class I mAb may reflect the recruitment of a higher percentage of T cells. The present study has shown for the first time that certain, but not all, the determinants of the HLA class I molecular complex are involved in the proliferation of T cells stimulated with the anti-CD2 mAb 9-1 + 9.6. Furthermore, the inhibitory effect of mAb CR11-351, KS1, Q6/64, and W6/32 on the proliferation of T cells stimulated with mAb OKT3 or with mAb BMA 031 indicates that the same determinants of HLA class I antigens play a differential regulatory role in T cell proliferation induced via the CD2 and CD3 pathway.     

Role of protein kinase C in the translational regulation of lipoprotein lipase in adipocytes

The hypertriglyceridemia of diabetes is accompanied by decreased lipoprotein lipase (LPL) activity in adipocytes. Although the mechanism for decreased LPL is not known, elevated glucose is known to increase diacylglycerol, which activates protein kinase C (PKC). To determine whether PKC is involved in the regulation of LPL, we studied the effect of 12-O-tetradecanoyl phorbol 13-acetate (TPA) on adipocytes. LPL activity was inhibited when TPA was added to cultures of 3T3-F442A and rat primary adipocytes. The inhibitory effect of TPA on LPL activity was observed after 6 h of treatment, and was observed at a concentration of 6 nM. 100 nM TPA yielded maximal (80%) inhibition of LPL. No stimulation of LPL occurred after short term addition of TPA to cultures. To determine whether TPA treatment of adipocytes decreased LPL synthesis, cells were labeled with [35S]methionine and LPL protein was immunoprecipitated. LPL synthetic rate decreased after 6 h of TPA treatment. Western blot analysis of cell lysates indicated a decrease in LPL mass after TPA treatment. Despite this decrease in LPL synthesis, there was no change in LPL mRNA in the TPA-treated cells. Long term treatment of cells with TPA is known to down-regulate PKC. To assess the involvement of the different PKC isoforms, Western blotting was performed. TPA treatment of 3T3-F442A adipocytes decreased PKC alpha, beta, delta, and epsilon isoforms, whereas PKC lambda, theta, zeta, micro, iota, and gamma remained unchanged or decreased minimally. To directly assess the effect of PKC inhibition, PKC inhibitors (calphostin C and staurosporine) were added to cultures. The PKC inhibitors inhibited LPL activity rapidly (within 60 min). Thus, activation of PKC did not increase LPL, but inhibition of PKC resulted in decreased LPL synthesis by inhibition of translation, indicating a constitutive role of PKC in LPL gene expression.