Maturation of cytolytic T lymphocytes

We have studied the maturation of cytotoxic T-lymphocytes (CTL) following primary and anamnestic responses in vivo and in vitro. Parameters evaluated included: frequency of effector CTL, specificity of binding to and lysis of target cells, killing and recycling ability of individual CTL, and the avidity of effector-target conjugation. While the frequency of effector CTL in the peritoneal cavity of BALB/c mice immunized against leukemia EL4 of C57BL/6 origin increases from 0 to 35% in 11 days of priming, a paradoxically lower frequency has been observed usually after 2 degrees and repeatedly after 3 degrees immunizations both in the peritoneal cavity and in the spleen. The H-2 haplotype and H-2 sub-loci specificity of CTL is preserved upon repeated immunizations. Likewise, the rate of killing and recycling of individual CTL do not change throughout immunizations, suggesting that the cytolytic activity of individual effector CTL is discrete ("quantal") and not subject to maturation upon repeated immunizations. On the other hand, inhibition of conjugate formation and of lysis by antibodies against target major histocompatibility complex (MHC) or effector Lyt-2 determinants is consistently less effective with 3 degrees CTL, suggesting an increase in avidity of effector/target interaction upon repeated immunizations. A striking increase in apparent avidity has been observed during CTL priming in mixed lymphocyte reaction, as deduced from blocking by target cell MHC antibodies. These results suggest that alloimmune CTL undergo maturation with respect to their ability to interact with the target, and that the composition of the responding population is subject to moderate selective processes driven by repeated antigenic stimuli.     

Stimulating effect of polyion on the production of cytolytic T lymphocytes in a cell culture in vitro

Introduction of polyion "vegetan" at the concentrations varying from 20 to 100 micrograms/ml into mixed lymphocyte culture (MLC) with insufficient antigenic stimulus, low immunogenic MLC, for the whole of the incubation period, leads to 30-70% increase in T-lymphocyte cytolytic activity. The effect of vegetan on the cytolytic activity of T-lymphocytes from normal MLC is less marked. In the in vivo model of primary synthesis of antibodies against heterologous erythrocyte antigens, vegetant causes 20-50-fold enhancement of a weak immune response compared to 1.5-2-fold when the latter approaches the highest levels. These findings indicate an inverse relationship between the immunostimulating activity of vegetan and the levels of the immune response, the latter being the target of activation. Vegetan was shown to induce more than 2-fold increase in the proliferation in both types of MLC as well as in mouse spleen lymphocyte monoculture. It is reasonable to propose that the preparation may stimulate the proliferation of both T-killers and other lymphocyte subpopulations.     

Histamine regulates the generation of human cytolytic T lymphocytes

Human cytolytic T lymphocytes (CTL) were generated in the presence and absence of histamine in order to define the role of this autacoid in immune regulation. Histamine (10(-8)-10(-4) M) suppressed the generation of class I specific CTL but, at 10(-4) M, actually increased class II specific cytolysis. Histamine acted at the level of CTL generation; histamine was not present in the cytolytic assay. When histamine was added to the cytolytic assay with CTL grown without histamine, the lytic ability of the effector cells was similar to that of controls. Histamine-induced suppression of class I specific cytolysis was blocked by continuous culture with the H2 antagonist ranitidine but not with the H1 antagonist pyrilamine. These data suggest that suppression was mediated by the H2 receptor. Continuous culture with histamine had no effect on T cell proliferation or the expression of cell surface molecules. Histamine-induced suppression of class I specific cytolysis was reversed by the addition of PHA to the cytotoxicity assay, showing that the cytolytic machinery was intact. These data provide evidence that histamine is involved in regulation of cytolytic T cells.     

Generation of cytolytic T lymphocytes in vitro. VIII. failure of anti-RS antisera to inhibit the generation of cytolytic T lymphocytes or cytolytic T lymphocyte activity.

Antibody reactive with "recognition structures" (RS) of mouse lymphoid cells for alloantigens (anti-RS) was prepared by immunization of F1 hybrid mice with parentalstrain lymphoid cells or with antibody produced in one parental strain against alloantigens of the other parental strain. Such antisera prevented generation of the "product of antigenic recognition" (PAR) that is produced within a few hours in cultures prepared with a mixture of lymphoid cells from genetically disparate mice. However, treatment of responding lymphoid cells with anti-RS sera and complement did not inhibit generation of cytolytic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC). Treatment of cells obtained from MLC with anti-RS sera and complement failed to inhibit cytolytic activity of such cells for specific alloantigens.     

Studies on the mechanism of suppression of primary cytotoxic responses by cloned cytotoxic T lymphocytes

We have shown in the accompanying companion paper that cloned cytotoxic T lymphocytes (CTL) can serve as veto cells in vitro, suppressing primary cytotoxic activity directed against antigens expressed by those cloned CTL but not against third party antigens. We now explore the mechanism of this antigen-specific suppression by cloned CTL, using as a model system the ability of G4, a BALB.B anti-H-2Dd CTL clone, to specifically suppress a primary in vitro anti-H-2b CTL response. G4 cells do not constitutively secrete a suppressor factor, because suppression cannot be mediated by supernatants removed from G4 cells at a time when they are routinely used as veto cells. Furthermore, medium removed from cultures suppressed by G4 will not suppress, indicating that the veto cell function of G4 is not mediated by soluble factors. Full suppression of primary anti-H-2b CTL responses requires that G4 be present throughout the 5-day mixed lymphocyte culture (MLC). Removal of G4 during the first 3 days of MLC results in a drastic reduction in the level of antigen-specific suppression, with a slight but reproducible loss of suppression after veto cell removal on day 4. The addition of G4 during the course of an ongoing MLC reveals that maximal suppression requires the presence of veto cells during the first 24 to 48 hr of culture. Thus, G4 cells must be present both early and late in an MLC to exert maximal veto cell suppression. Several experiments suggest that G4-induced veto cell activity is unlikely to be due to cytolysis of CTL precursors which are capable of recognizing G4. G4 cannot specifically recognize these CTL precursors, and G4 cells are inefficient at lectin-mediated lysis of non-tumor cell targets. Furthermore, we show that G4 cells cannot lyse CTL which recognize them. Finally, dilutions of anti-clonotypic antibodies which completely block both lectin-mediated and specific cytolysis by G4 do not block (and in fact enhance) G4-mediated veto cell activity.     

Accessory cell requirements for the generation of cytolytic T lymphocytes.

A system is presented that may simplify the study of accessory cell requirements for CTL generation. Cortisone resistant (CR) thymocytes containing alloreactive CTL precursors do not respond to allogeneic tumor cells unless non-T accessory cells are added to culture. In addition, splenic T cells do not respond to allogeneic tumor cells in the absence of non-T accessory cells. These accessory cells share several properties of macrophages.