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.     

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.     

Inhibition by anti-HLA class II monoclonal antibodies of monoclonal antibody OKT3-induced T cell proliferation. Studies at the mRNA level

mAb to monomorphic determinants of HLA class II Ag have been shown to inhibit monocyte-dependent OKT3-induced T cell proliferation, indicating that MHC class II molecules play a regulatory role also in Ag nonrestricted, CD3-induced T cell proliferation. This effect involves several steps in the process of T cell activation and proliferation, including IL-1 beta, IL-6, and IL-2 secretion and IL-2R alpha expression. In the present study, we analyzed the effect of an anti-HLA class II mAb (Q5/6) on the mRNA expression of genes related to monocyte and T cell activation. mRNA levels for early (early c-myc, c-fos) and late (late c-myc, N-ras, c-myb) genes involved in T cell activation were determined as well as mRNA levels for IL-1 beta, IL-6, IFN-gamma, IL-2, and IL-2R alpha. The kinetics of mRNA induction for ICAM-1 was also investigated. The results show that in T lymphocytes the expression of c-fos and early c-myc mRNA was unaffected by mAb Q5/6, whereas the c-myb and N-ras mRNA levels were strongly diminished as well as those of IL-2, IL-2R alpha, and IFN-gamma mRNA. An early increase of ICAM-1 mRNA was partially inhibited. In monocytes, a marked reduction of IL-1 beta and IL-6 mRNA was found. It is concluded that the HLA class II determinant involved in the inhibition mechanism can be engaged in the control of IL-1 beta and IL-6 mRNA levels and constitute an accessory signal up-regulating IL-2 and IL-2R alpha gene activation, through a pathway not affecting c-myc and c-fos expression.     

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.     

A polymorphic monoclonal anti-BOLA class I antibody

Cytotoxic monoclonal antibody IVA 44 was generated after the intraperitoneal immunization with peripheral blood mononuclear (PBM) cells and the boost by the intrasplenic inoculation of skin graft. The detected membrane antigen isolated by immunoprecipitation appears to be composed of two subunits characteristic for the MHC class I molecules. The antibody IVA 44 exhibited a different reactivity: it recognized the BoLA A14 (A8) specificity in animals typed in the Fifth BoLA workshop, while it reacted with all A8 positive animals including subtypes A14 and A15 in Czech and Slovak cattle. It is concluded that mAb IVA 44 might detect the broad subtype of A8 covering A14 and certain A15 split(s). The diverse A15 reactivity of this mAb in the workshop and our population could be explained by the different occurrence of A15 splits in both populations.     

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.     

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.     

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.     

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.     

A bovine monoclonal antibody detecting a class I BoLA antigen.

The bovine IgG1 monoclonal antibody (mAb) ILA70 was made by immunizing a calf with peripheral blood mononuclear cells (PBM) from a BoLA-w10 homozygous heifer and subsequently fusing lymphocytes from the local lymph-node with the heterohybridoma 53B3. Family and population studies, antibody binding inhibition and immunoprecipitation of the target antigen all indicate that ILA70 detects a polymorphic epitope on a bovine class I major histocompatibility complex (MHC) molecule. The antibody is complement fixing and so may be used in a standard cytotoxicity assay. Ascitic fluid with antibody activity many times greater than that of the tissue-culture supernatant has been prepared in nude mice. The antibody-producing heterohybridoma has been subcloned three times and appears to be stable. Such heterohybridomas may prove to be a valuable source of particularly discriminating and informative mAbs for the serological analysis of the products of the bovine MHC.     

Reactivity of lymphocytes to a progesterone receptor-specific monoclonal antibody

In this study we present evidence for reactivity of pregnancy lymphocytes, but not nonpregnancy lymphocytes, with the progesterone receptor-specific monoclonal antibody mPRI. Using an avidin-biotin peroxidase detection system, we found a nuclear staining in 14.6 +/- 3.7% (mean +/- SEM, N = 27) of pregnancy lymphocytes, while only 0.47 +/- 0.33% (mean +/- SEM, N = 15) of nonpregnancy lymphocytes reacted with the antibody. To characterize the receptor-bearing subset, CD8+ and CD4+ cells were depleted by complement-dependent lysis. Depletion of CD8+ cells was accompanied by 62 +/- 18% loss of progesterone receptor-bearing cells, while depletion of CD4+ cells resulted in a twofold increase in the number of positively staining lymphocytes. In nonpregnancy lymphocytes a 3-day PHA treatment, as well as allogeneic stimulation, resulted in a significant increase in the number of receptor-containing cells. These results suggest that pregnancy, but not nonpregnancy, lymphocytes contain progesterone binding structures, and that these are inducible by mitogenic or alloantigenic stimuli.     

Signal transduction through class I MHC by a monoclonal antibody that detects multiple murine and human class I molecules.

We have generated a hamster anti-mouse class I reactive mAb that is capable of activating T cells in the presence of the cofactor PMA, as assayed by both IFN-gamma production and cellular proliferation. This mAb detects an epitope present on the majority of murine class I molecules, with the known exceptions of H-2Kk and H-2Kq, and is therefore not beta 2-microglobulin-specific. It also recognizes multiple human class I molecules. The epitope recognized by this antibody maps to the class I alpha 1 domain. The activation properties of this mAb are not mediated exclusively through the glycosylphosphatidylinositol-linked Qa-2 molecule, as the antibody activates spleen cells from Qa-2 negative strains. Although class I molecules are not usually considered as activation Ag, these data demonstrate their potential for involvement in signal transduction.     

Stimulation of human T lymphocytes by PHA-activated autologous T lymphocytes: Analysis of the role of Ia-like antigens with monoclonal antibodies

Human T lymphocytes activated with PHA express Ia-like antigens and acquire the ability to stimulate autologous T lymphocytes in mixed lymphocyte reaction. This reaction is immunological in nature since it has specificity and memory. Ia-like antigens play a role in the stimulation of T lymphocytes by autologous PHA-T lymphocytes since monoclonal antibodies to Ia-like antigens can significantly, although not completely, inhibit the stimulation.     

Anti-HLA class I antibodies inhibit the T cell-independent proliferation of human B lymphocytes

The role of major histocompatibility complex-encoded class I molecules in the proliferation of human B lymphocytes is presently unclear. This question was addressed by investigating the effect of three individually derived anti-HLA class I monoclonal antibodies (mAb) on purified human B cells (less than 1.5% T cells) stimulated by either the T-independent mitogen Staphylococcus aureus or the phorbol ester, phorbol-12-myristate-13-acetate. The three anti-HLA class I antibodies, whether specific for gene products of the HLA-A locus (mAb 131), HLA-B locus (mAb 4E), or HLA-A, -B, and -C locus (mAb W6/32), inhibited S. aureus-induced proliferation by 70 to 90%. This inhibition was significant over a 5-day culture period, was not altered by the addition of exogenous interleukin 2 or B cell growth factor, and was not due to nonspecific cytotoxicity. In addition, the inhibition of proliferation was unchanged when the mAb were added 12 hr after the initiation of culture. The proliferative response was not affected by either of the control antibodies OKB7 and R3-367. In contrast with S. aureus-stimulated B cells, phorbol-12-myristate-13-acetate-induced proliferation was resistant to the inhibitory activity of HLA class I-specific antibodies. These results suggest that HLA class I molecules are involved in human B lymphocyte proliferation and may regulate a critical event preceding the upregulation of protein kinase C activity.     

Thromboxane A2 fails to induce proliferation of smooth muscle cells enriched with eicosapentaenoic acid and docosahexaenoic acid.

Thromboxane A2 (TXA2) released from aggregating platelets and injured vessel wall stimulates smooth muscle cell proliferation, which may contribute to the development of vascular lesion formation after percutaneous transluminal coronary angioplasty. Polyunsaturated fatty acids (n-3) present in the fish oils have been shown to have anti-atherosclerotic effects. In view of this, we examined the effect of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the active ingredients of fish oils on TXA2 induced smooth muscle cell proliferation. To find out the specificity of these fatty acids we used gamma-linolenic acid (n-6) and oleic acid (n-9) as controls. It was found that TXA2 failed to stimulate proliferation of smooth muscle cells preloaded with EPA or DHA but not with gamma-linolenic acid or oleic acid. Further, when smooth muscle cells were preloaded with both EPA and DHA, they acted together in preventing the TXA2 induced smooth muscle cell proliferation. These results demonstrate that one of the mechanisms by which fish oils may prevent neointima formation is by making smooth muscle cells less responsive to TXA2 induced proliferation of smooth muscle cells.     

Induction of human T cell proliferation by a monoclonal antibody to CD5.

A mouse mAb, TS 43, which recognized the human CD5 molecule, was found to induce the proliferation of human peripheral blood T cells. TS 43 mAb precipitated from 125I-radiolabeled T cells a 67-kDa band, which comigrated with the 67-kDa band precipitated by the anti-CD5 mAb OKT1. Preclearing of cell lysates with OKT1 mAb abolished the capacity of TS 43 mAb to precipitate radiolabeled material from T cell lysates. Furthermore, a mouse T cell hybridoma transfected with human CD5 was stained by TS 43 mAb. T cell proliferation mediated by TS 43 mAb was monocyte dependent, and was accompanied by IL-2R expression and by IL-2 synthesis. T cell activation by TS 43 mAb involved a rise in intracellular calcium level (CA2+)i and was dependent on the expression of the TCR/CD3 complex because no rise in (Ca2+)i was observed in a TCR-beta-deficient Jurkat T cell mutant. This study indicates that CD5 should be added to the list of surface molecules that can signal T cells to proliferate.