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Anti-HLA class I monoclonal antibody 01.65 inhibits the proliferative response of PHA-activated human T lymphocytes from peripheral blood mononuclear cells. The recruitment rate in the cell cycle is slack and the G1 and S phases are prolonged. Among the early events after PHA activation, only the calcium-dependent PKC activity appears to be modified: particulate PKC is completely depleted while cytosolic residual PKC is reduced by 80% after MAb 01.65 treatment. We have carried out in greater detail the study of c-myc gene regulation by MAb 01.65 and the results are as follows: (i) c-myc RNA transcription is normally initiated and finished, suggesting a post-transcriptional regulation of c-myc gene expression; (ii) no alteration in c-myc mRNA stability has been documented; (iii) steady-state levels of c-myc mRNA expression by Northern blot analysis and PCR amplification are decreased in the cytoplasmic compartment, while in the nuclear compartment they appear to be increased. Nuclear accumulation of mature mRNA after MAb 01.65 and PKC inhibitor (H7 and StSp) treatment appears to be the most probable mechanism involved. The possible implications of this are discussed.

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.

Anti-HLA class I monoclonal antibody (mAb) 01.65 inhibited phytohaemagglutinin (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.

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.

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.

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.

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.

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.