Publisher Summary Studies of cytotoxicity by human lymphocytes revealed not only that both allogeneic and syngeneic tumor cells were lysed in a non-MHC-restricted fashion, but also that lymphocytes from normal donors were often cytotoxic. Lymphocytes from any healthy donor, as well as peripheral blood and spleen lymphocytes from several experimental animals, in the absence of known or deliberate sensitization, were found to be spontaneously cytotoxic in vitro for some normal fresh cells, most cultured cell lines, immature hematopoietic cells, and tumor cells. This type of nonadaptive, non-MHC-restricted cellmediated cytotoxicity was defined as “natural” cytotoxicity, and the effector cells mediating natural cytotoxicity were functionally defined as natural killer (NK) cells. The existence of NK cells has prompted a reinterpretation of both the studies of specific cytotoxicity against spontaneous human tumors and the theory of immune surveillance, at least in its most restrictive interpretation. Unlike cytotoxic T cells, NK cells cannot be demonstrated to have clonally distributed specificity, restriction for MHC products at the target cell surface, or immunological memory. NK cells cannot yet be formally assigned to a single lineage based on the definitive identification of a stem cell, a distinct anatomical location of maturation, or unique genotypic rearrangements.

Biology of natural killer cells.

Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20

Publisher Summary This chapter focuses on the important discovery that virus-specific cytotoxic T cells are dually specific for virus and for a self cell surface antigen encoded by the major histocompatibility complex (MHC). The initial work was carried out on the lymphocytic choriomeningitis virus system but it soon became evident that the same phenomenon applied to many other viruses. In addition, the same principle has been found to hold for other antigenic systems, such as trinitrophenyl coupled to cells, minor histocompatibility antigens, and the H-Y model. Graft rejection and the need for genetically homogeneous inbred mouse strains for cancer research led to the development of transplantation immunology and immunogenetics. The result is that the gene complex coding for major transplantation antigens is one of the better understood mammalian genetic regions. Cytotoxic T-cell specificity is comparable to serological specificity. Because quantification of specificity or cross-reactivity is difficult, and because of the technical limitations of these cytotoxic T-cell assays, results are interpreted with great reservation. MHC restriction reflects the fact that the effector function of T cells is determined by the kind of Self-H recognized together with the foreign antigen on cell surfaces: K and D are receptors for lytic signals, I determinants are receptors for cell differentiation signals that are delivered antigen-specifically by T cells.

MHC-restricted cytotoxic T cells: studies on the biological role of polymorphic major transplantation antigens determining T-cell restriction-specificity, function, and responsiveness.

Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation

MANY investigators have been successful in the maintenance of long term tissue culture of human bone marrow-derived (B) cells. These cell lines have been established from both normal subjects1 and from patients with lymphoproliferative disorders2. In most cases, long term B-cell lines have been shown to harbour the Epstein–Barr virus genome which some investigators feel is required for establishment and maintenance of long-term cultures3. There are fewer reports describing continuous culture of human thymus derived (T) cell lines, and when successful, the lines have only been established from patients with acute lymphocytic leukaemia4. Although these cell lines have been shown to bear surface markers of normal human T lymphocytes, there have been no reports which suggest that they possess the ability to respond to immunologic stimuli or to differentiate into antigen-specific lymphocytes. Cytotoxic murine T cells have been kept in continuous culture only through repetitive mixed-lymphocyte stimulation5. In contrast to long term human lymphocyte lines, these cells proliferated only when stimulated with allogeneic lymphocytes and eventually died after a few weeks in culture. Morgan, Ruscetti and Gallo recently reported a method by which medium conditioned by phytohaemagglutinin-stimulated normal human lymphocytes allowed for the selective long-term growth of normal T cells6. In contrast to the previously mentioned cell lines, the proliferation of these reported T-cell cultures was totally dependent on the presence of an exogenously-produced growth factor supplied by the conditioned medium. In this report we describe an adaptation of this method which allows the long term culture of antigen-selected cytotoxic T cells which continue to demonstrate high levels of syngeneic tumour-specific cytotoxicity after more than 4 months in culture.

Long term culture of tumour-specific cytotoxic T cells.

The in vitro cytotoxic effect of spleen cells of mice immunized by tumour allografts was studied by measuring target cell inactivation as a function of release of radioactive label (51Cr) or loss of cloning efficiency. When sensitized lymphoid cells were incubated with target cells at a ratio of 100:1, up to 90 per cent of the incorporated label was released within 6–9 hours, while the number of clone-forming cells was reduced by up to 99 per cent in the same time period. Isoantiserum from the graft recipients, as well as its 19S and 7S fractions, protected target cells against the toxic effect of the spleen cells, but a lipoprotein antigen isolated from the tumour cells failed to inhibit the cytotoxic reaction. Target cell lysis as measured by specific release of 51Cr was partially inhibited by actinomycin-D and by cycloheximide at concentrations which effectively blocked DNA-dependent RNA and protein synthesis.

Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs.

Cellular immunity induced by tumour allografts in inbred mice was studied with the help of an in vitro assay system measuring the cytotoxic effect of sensitized lymphocytes on 51Cr-labelled target cells. It is shown that lymphoid cells from spleen, lymph nodes and blood of the allograft recipients reach a peak of cytotoxic activity on days 10–11 after immunization. Incubated with labelled target cells at a ratio of 100:1, the sensitized lymphocytes caused the specific release of up to 70 per cent of the radioactivity within 1 hour. A second population of target cells added to the same cell suspension was destroyed at a slightly accelerated rate, suggesting stimulation of the effector cells by the first interaction. The cytotoxic activity of circulating lymphocytes was found to reach a plateau between days 20 and 60 after immunization, while the activity of spleen cells dropped to low levels in the same time period. The specificity of target cell destruction by sensitized lymphocytes is demonstrated by the lack of lytic activity for syngeneic target cells, and by the selective destruction of target cells carrying a tumour specific antigen. Tumour cells, lymphocytes and embryonic fibroblasts of the donor strain are shown to differ considerably in their sensitivity to lysis by immune lymphocytes.

Studies of allograft immunity in mice. I. Induction, development and in vitro assay of cellular immunity.

The mechanisms of the in vitro interaction of sensitized lymphocytes and allogeneic target cells has been studied in a tumour allograft system in inbred mice The cytotoxic effect of sensitized lymphocytes is shown to require the presence of Ca+ + and Mg+ + Pretreatment of the lymphocytes with trypsin led to inhibition of cytotoxicity, followed by spontaneous reversal after 1–3 hours incubation Reactivation was found to be blocked by an inhibitor of protein synthesis (cycloheximide) Cortisone was not found to inhibit the lytic interaction significantly; an occasional effect is thought to be due to toxicity of cortisone for lymphocytes as revealed by dye exclusion test Inhibition of DNA-synthesis with FUdR (an inhibitor of the enzyme thymidine synthetase) did not reduce the lytic activity of sensitized lymphocytes

Isologous anti-target cell sera induced in various strains of inbred mice were found to be ineffective in blocking the cellular immune reaction in vitro when directed against a minor part of the antigenic complex, but strongly inhibitory when reactive against a major part or the whole complex Similarly, target cells lacking several of the sensitizing H-2 antigens were not lysed An isologous anti-lymphocytic serum induced in the graft donor strain and directed against the recipient strain (lymphocyte donor) did not inhibit the cytotoxic reaction In a heterologous system on the other hand, the lytic effect of guinea-pig lymphocytes sensitized against mouse target cells was effectively blocked by an anti-lymphocytic serum induced in mice of the graft donor strain by injection of recipient (guinea-pig) spleen cells

Studies of allograft immunity in mice: II. Mechanism of target cell inactivation in vitro by sensitized lymphocytes

Quantitative Assay oftheLytic Action ofImmune Lymphoid Cells on5"Cr-Labelled Allogeneic Target Cells Invitro; Inhibition byIsoantibody andbyDrugs

Summary.Themechanisms oftheinvitro interaction ofsensitized lymphocytes andallogeneic target cells hasbeenstudied inatumourallograft system ininbred mice.Thecytotoxic effect ofsensitized lymphocytes isshowntorequire the presence ofCa++andMg++.Pretreatment ofthelymphocytes withtrypsin ledto inhibition ofcytotoxicity, followed byspontaneous reversal after 1-3hours incubation. Reactivation wasfoundtobeblocked byaninhibitor ofprotein synthesis (cycloheximide). Cortisone wasnotfoundtoinhibit thelytic interaction significantly; anoccasional effect isthought tobeduetotoxicity ofcortisone forlymphocytes asrevealed bydyeexclusion test. Inhibition ofDNA-synthesis withFUdR (aninhibitor oftheenzymethymidine synthetase) didnotreducethelytic activity ofsensitized lymphocytes. Isologous anti-target cell serainduced invarious strains ofinbred micewere foundtobeineffective inblocking thecellular immunereaction invitro when directed against aminorpartoftheantigenic complex, butstrongly inhibitory whenreactive against amajorpartorthewholecomplex. Similarly, target cells lacking several ofthesensitizing H-2antigens werenotlysed. Anisologous antilymphocytic seruminduced inthegraft donorstrain anddirected against the recipient strain (lymphocyte donor) didnotinhibit thecytotoxic reaction. Ina heterologous system ontheother hand, thelytic effect ofguinea-pig lymphocytes sensitized against mousetarget cells waseffectively blocked byananti-lymphocytic seruminduced inmiceofthegraft donorstrain byinjection ofrecipient (guinea-pig) spleen cells.

K. T. Brunner

Papers

Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs.

Studies of allograft immunity in mice. I. Induction, development and in vitro assay of cellular immunity.

Studies of allograft immunity in mice: II. Mechanism of target cell inactivation in vitro by sensitized lymphocytes

Quantitative Assay oftheLytic Action ofImmune Lymphoid Cells on5"Cr-Labelled Allogeneic Target Cells Invitro; Inhibition byIsoantibody andbyDrugs

Studies ofAllograft Immunity inMice II.MECHANISM OF TARGET CELLINACTIVATIONInVitro BYSENSITIZED LYMPHOCYTES