Purification and characterization of a T cell specific serine proteinase (TSP-1) from cloned cytolytic T lymphocytes.

We describe the purification of a T cell specific serine proteinase derived from a cloned murine cytolytic T lymphocyte line. Analysis of the enzyme by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a mol. wt of approximately 60 kd under non-reducing conditions and of approximately 30 kd under reducing conditions. The proteinase cleaves the model peptide substrate H-D-Pro-Phe-Arg-NA, at the 4 nitroanilide (NA) group with high efficiency. Much lower or no activity of the enzyme is found against synthetic peptide substrates carrying other amino acid (AA) sequences at position P2, P3 adjacent to L-arginine or against substrates in which AA other than L-arginine are bound to the NA group. Optimal pH for the cleavage of H-D-Pro-Phe-Arg-NA is in the range of 8.0-8.5. The enzyme is strongly inhibited by inhibitors of serine proteinases, diisopropylfluorophosphate, phenylmethane-sulfonyl fluoride, m-aminobenzamidine, aprotinin, and leupeptin but not by inhibitors of either thiol-, metallo- or carboxyl-proteinases. We propose the designation TSP-1 (T-cell derived serine proteinase 1) for this enzyme. TSP-1 has the capacity to stimulate B lymphocytes for proliferation in the absence of antigen.     

Generation of IL-2-dependent cytolytic T lymphocytes (CTLs) with altered TCR responses derived from antigen-dependent CTL clones.

Ag-specific CD8+ CTL clones require TCR stimulation to respond to IL-2 for growth. Because IL-2 may be produced in the vicinity of CD8+ CTLs when Ag is limiting at the end of an immune response, we have examined the effect of culturing viral-specific CTL clones in IL-2 in the absence of antigenic stimulation. Limiting dilution analysis revealed a high precursor frequency for CTL clones derived from IL-2 propagation (termed CTL-factor dependent (FD)) that are dependent upon exogenous IL-2 for growth and survival and no longer require TCR stimulation to proliferate. Culturing CTL-FDs with infected splenocytes presenting Ag and IL-2 did not revert the clones but did lead to a TCR-induced inhibition of proliferation. The derived CTL-FDs have lost the ability to kill via the perforin/granule exocytosis mechanism of killing, although they express similar levels of TCR, CD3epsilon, CD8alphabeta, CD45, and LFA-1 compared with the parental clones. The CTL-FDs retain Fas ligand/Fas-mediated cytotoxicity, and IFN-gamma production and regulate the expression of CD69 and IL-2Ralpha when triggered through the TCR. A parental CTL protected BALB/c mice from a lethal challenge of influenza virus, whereas a CTL-FD did not. These findings represent a novel regulatory function of IL-2 in vitro that, if functional in vivo, may serve to down-regulate cellular immune responses.     

Effects of cytotoxic T lymphocytes (CTL) directed against a single simian immunodeficiency virus (SIV) Gag CTL epitope on the course of SIVmac239 infection

Vaccine-induced cytotoxic T lymphocytes (CTL) have been implicated in the control of virus replication in simian immunodeficiency virus (SIV)-challenged and simian-human immunodeficiency virus-challenged macaques. Therefore, we wanted to test the impact that vaccine-induced CTL responses against an immunodominant Gag epitope might have in the absence of other immune responses. By themselves, these strong CTL responses failed to control SIVmac239 replication.     

The protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) inhibits PMA-induced promiscuous cytolytic activity but not specific cytolytic activity by a cloned cytolytic T lymphocyte

Phorbol 12-myristate 13-acetate (PMA) induces the cytolytic T lymphocyte (CTL) clone 4D (H-2b anti-H-2d) to promiscuously kill the inappropriate target EL-4 (H-2b). The protein kinase C (PKC) inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) inhibited the PMA-induced promiscuous lympholysis. The concentration of H-7 that inhibited PMA-induced lympholysis by 50% (IC50) was calculated to be 4 microM, which closely approximates the reported IC50 of H-7 of 6 microM for PKC activity in vitro. In striking contrast, specific cytolysis of appropriate P815 (H-2d) target cell by CTL clone 4D was not inhibited by concentrations of H-7 which inhibited PMA-induced promiscuous lympholysis. These results indicate that PMA-induced promiscuous lympholysis of inappropriate target cell is triggered via activation of PKC, whereas PKC activation is not obligatory in triggering CTL clone 4D to specifically kill appropriate target cells. Thus, these data suggest that cloned CTL have two or more triggering mechanisms than may initiate one or more cytolytic pathways.