Search

A T4+ proliferative, noncytotoxic cloned line acquires specific lytic function by treatment with recombinant interferon alpha or gamma. Simultaneous with the acquisition of this new cell function, a rearrangement of the T-cell receptor alpha gene occurs. These changes necessitate a revised concept of a T-cell clone regarding its T-cell receptor gene configuration and cell function.

T-cell receptor genes are found in the germ-line configuration as well as in rearranged forms, as detected in T lymphocytes by different patterns in Southern blot analysis. We have recently shown that cytotoxic T-lymphocyte (CTL) "precursor" clones, in which rearrangements are not detected in the gene encoding the T-cell receptor alpha-chain (TCRA), acquire specific lytic function induced by treatment with recombinant interferon alpha or gamma. We now report that, coincident with the acquisition of cytotoxic function by the precursor CTL clone; recombinant interferon alpha appears to induce a rearrangement of TCRA. In addition, in a mature CTL clone (i.e., one already showing lytic function) in which one TCRA allele is rearranged, treatment with recombinant interferon alpha appears to induce a new rearrangement of a TCRA gene.

Allostimulated T lymphocytes were cloned by micromanipulation and expanded in IL-2 conditioned medium. Three T3+,T4+,T8-, clones called BJ1, BJ4, and BJ37, were extensively studied. The BJ1 cells were able to proliferate and kill the specific target. The BJ4 and BJ37 cells were able to proliferate with the specific restimulator but could not kill even in lectin-dependent cell-mediated cytotoxic assay; however, they acquired the specific cytolytic activity in the 6-day culture when fresh irradiated autologous peripheral blood mononuclear cells as feeder cells were added to the specific irradiated Epstein-Barr virus transformed cell line, in the presence of recombinant IL-2. This observation strongly suggested that the culture conditions could be involved in the differentiation of proliferative clones into cytotoxic T lymphocyte (CTL) clones, by the lymphokines, either present in the IL-2 conditioned medium or secreted by the mixed allogeneic irradiated feeder cells. Moreover, it was shown that the acquisition of the cytolytic function could be blocked by the monoclonal antibody LeoA1, previously described and which recognized the TLiSA1 structure involved in the CTL differentiation.

Six-day allosensitized human peripheral blood lymphocytes treated for 12-18 hr with phorbol 12-myristate 13-acetate (PMA) were found to lose the capacity to kill the specific target in a standard cell-mediated lympholysis (CML) assay, but they were still effective in their ability to kill the tumor cell line K562. We investigated which antigens on the cell membrane involved in alloimmune recognition might be modified by PMA, since it was found that in a lectin-dependent CML assay, the lytic mechanism was not impaired. For this purpose, T3+, T4+ allospecific cytotoxic T lymphocyte (CTL) clones and either T3+ or T3- natural killer (NK) clones were generated from 6-day allostimulated lymphocytes. The results indicated that PMA inhibited the cytolytic function of both alloimmune CTL and NK T3+ clones. In contrast, PMA did not modify the effector cell function of T3- NK clones. Phenotypic analysis of T-cell surface antigens from T3+ clones showed that T3 molecule expression on the cell membrane was reduced by 80-90% after PMA treatment, whereas expression of both accessory T4 molecules, involved in antigen recognition, and receptor for interleukin 2 was increased. Moreover, the loss of function was transitory and could be restored 4 days after PMA treatment when the T3 molecules were fully reexpressed at the cell surface. Taken together, these data strongly suggest that (i) PMA prevents cell-mediated cytotoxicity by modulating the disulfide-linked heterodimer associated to T3 and described as the receptor for antigen on the cell surface of major histocompatibility complex (MHC) and non-MHC specific CTL clones, without affecting the lytic mechanism per se, and (ii) the expression of the receptor for the antigen present on the tumor cell line K562 is not decreased on T3- NK clones after PMA treatment and must be different from that on T3+ T-cell clones.

Human cloned antigen-specific T lymphocytes were used to characterize DNA rearrangements using a cDNA probe from the T cell receptor (TCR)-alpha chain gene. Rearranged patterns were detected in some T cell clones, confirming that normal mature T cells are rearranged in TCR-alpha locus. Similarly, rearranged DNA patterns were found in T cell clones from the same panel, using a DNA probe (clone K-40, [1]) isolated from chromosome 14 (14q11), where the TCR-alpha locus has been mapped. These results suggest that this genomic DNA clone is located within the TCR-alpha chain locus.

Experiments were carried out to determine the effect of urea and creatinine on the solubility of nitrofurantoin in water at different temperature and pH conditions. The addition of urea to aqueous media increased nitrofurantoin solubility up to a maximum concentration level and then decreased solubility at higher urea concentrations. The amount of urea needed to bring about maximum nitrofurantoin solubility was dependent on temperature and ranged between 1.75 and 2.50%. Spectral studies suggest a possible interaction between urea and nitrofurantoin molecules. Nitrofurantoin solubility increased with an increasing creatinine concentration ranging from 0.05 to 1.6%. Spectral studies indicate a strong interaction between creatinine and nitrofurantoin molecules in solution. The combined effect of urea and creatinine of the solubility of nitrofurantoin could account for the absence of crystalluria with this drug, even though unusually high concentrations in urine have been reported.