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.

Four previously healthy homosexual men contracted Pneumocystis carinii pneumonia, extensive mucosal candidiasis, and multiple viral infections. In three of the patients these infections followed prolonged fevers of unknown origin. In all four cytomegalovirus was recovered from secretions. Kaposi's sarcoma developed in one patient eight months after he presented with esophageal candidiasis. All patients were anergic and lymphopenic; they had no lymphocyte proliferative responses to soluble antigens, and their responses to phytohemagglutinin were markedly reduced. Monoclonal-antibody analysis of peripheral-blood T-cell subpopulations revealed virtual elimination of the Leu-3 / helper/inducer subset, an increased percentage of the Leu-2 + suppressor/cytoxic subset, and an increased percentage of cells bearing the thymocyte-associated antigen T10. The inversion of the T/ helper to suppressor/cytotoxic ratio suggested that cytomegalovirus infection was an important factor in the pathogenesis of the immunodeficient state. A high level of exposure of male homosexuals to cytomegalovirus-infected secretions may account for the occurrence of this immune deficiency.

Pneumocystis carinii pneumonia and mucosal candidiasis in previously healthy homosexual men: evidence of a new acquired cellular immunodeficiency.

Activation in lectin-free interleukin 2 (IL-2) containing supernatants of peripheral blood mononuclear leukocytes (PBL) from cancer patients or normal individuals resulted in expression of cytotoxicity toward 20 of 21 natural killer (NK)-resistant fresh solid tumor cells tested. Fresh solid tumor cells were resistant to NK-mediated lysis in 10 autologous patients' PBL-tumor interactions, and from 17 normal individuals tested against 13 allogeneic fresh tumors. Culture of PBL in IL-2 for 2-3 d was required for the lymphokine activated killers (LAK) to be expressed, and lytic activity toward a variety of NK-resistant fresh and cultured tumor targets developed in parallel. Autologous IL-2 was functional in LAK activation, as well as interferon-depleted IL-2 preparations. Irradiation of responder PBL before culture in IL-2 prevented LAK development. Precursors of LAK were present in PBL depleted of adherent cells and in NK-void thoracic duct lymphocytes, suggesting that the precursor is neither a monocyte nor an NK cell. LAK effectors expressed the serologically defined T cell markers of OKT.3, Leu-1, and 4F2, but did not express the monocyte/NK marker OKM-1. Lysis of autologous fresh solid tumors by LAK from cancer patients' PBL was demonstrated in 85% of the patient-fresh tumor combinations. Our data present evidence that the LAK system is a phenomenon distinct from either NK or CTL systems that probably accounts for a large number of reported nonclassical cytotoxicities. The biological role of LAK cells is not yet known, although it is suggested that these cells may be functional in immune surveillance against human solid tumors.

https://rupress.org/jem/article-pdf/155/6/1823/1092516/1823.pdf

Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes.

The expression of the Hodgkin's disease associated antigen Ki-1 in reactive and neoplastic lymphoid tissue: evidence that Reed-Sternberg cells and histiocytic malignancies are derived from activated lymphoid cells.

A series of monoclonal antibodies was used to define three discrete stages of human intrathymic T-cell differentiation The earliest stage was confined to <10% of thymocytes, which were·reactive with both OKT9 and OKT10 Subsequently, approximately 70% of human thymocytes acquired a thymocyte-restricted antigen, OKT6, lost OKT9 antigen, and expressed reactivity with OKT4 and OKT5 These last two monoclonal antibodies were previously shown to define inducer (helper) and cytotoxic/suppressor populations, respectively, in peripheral blood The OKT4+, OKT5+, OKT6+ “common” thymocyte population represents the majority of thymocytes and accounts for more than 70% of thymocytes With further maturation, thymocytes lose OKT6 reactivity, segregate into OKT4+ and OKT5+ subsets, and acquire reactivity with OKT3 (and OKT1) This latter stage corresponds to the more functionally mature subset The possible relationship of acute lymphoblastic leukemia of T-cell lineage to these proposed stages of intrathymic differentiation was determined Analysis of 25 tumor populations showed that 21 could be related to one or another differentiative stage The majority (15/21) were derived from an early thymocyte or prothymocyte subpopulation, 5/25 were derived from a common thymocyte subpopulation, and 1/25 was derived from a mature (OKT3+) subpopulation These data suggest that is it now possible to define stages of T-cell differentiation that can be related to T-cell malignancies in humans

https://www.pnas.org/content/pnas/77/3/1588.full.pdf

Discrete stages of human intrathymic differentiation: Analysis of normal thymocytes and leukemic lymphoblasts of T-cell lineage

A monoclonal antibody directed at a determinant on human peripheral blood monocytes was produced and characterized. This hybridoma antibody, termed OKM1, was reactive by indirect immunofluorescence and complement- (C) mediated lysis with adherent mononuclear cells. OKM1 was unreactive with lymphocytes, thymocytes, lymphoblastoid cell lines, and tumor cells of the T or B cell lineage. In contrast, acute myelomonocytic leukemia cells and granulocytes were reactive with the antibody. Pretreatment of peripheral blood mononuclear cells with OKM1 and C before culture with soluble antigens totally abolished their antigen-induced proliferative response. This function was restored by addition of 1% adherent cells. These findings provided additional support for the notion that OKM1 was reactive with monocytes. In addition, OKM1 appeared to define two distinct populations of monocytes; an adherent population of large cells bearing surface Ia determinants and a nonadherent population of small, Ia-negative cells. These OKM1+ Ia- cells were found to be a contaminant of most fractionated mononuclear cell subsets including the E-SIg-Null cell population.

A monoclonal antibody reactive with human peripheral blood monocytes.

A hybridoma-secreting monoclonal antibody was produced from the spleen cells of a mouse immunized with human thymocytes. This hybridoma antibody, termed OKT5, was reactive by indirect immunofluorescence with 80% of human thymocytes but only 20% of peripheral blood T cells. Moreover, OKT5 was unreactive with normal B cells, null cells, and macrophages at any dilution tested. A similar pattern of reactivity was seen with an equine antiserum to human thymocytes termed anti-TH2. Fluorescence-activated cell sorting demonstrated that the OKT5 antibody reactivity on peripheral T cells was restricted to the majority of the previously defined TH2+ subpopulation. In functional studies, the OKT5+ subset, like the TH2+ subset, proliferated well to the mitogen Con A and to alloantigens, and contained cytotoxic effector cells after sensitization in MLC, and suppressor effector cells after activation with Con A. In addition, like the TH2+ T cell, the OKT+ T cell was virtually unresponsive to soluble antigen. Thus, the OKT5 monoclonal antibody is reactive with the cytotoxic/suppressor T cell subset. OKT5 should provide an important probe to assess the status of suppressor cells in human disease.

A monoclonal antibody reactive with the human cytotoxic/suppressor T cell subset previously defined by a heteroantiserum termed TH2.

The nature of Ia antigens which appear on human T cells after activation and the stimuli required for their expression was examined utilizing a monoclonal antibody reactive with the Ia antigen framework. T cells were purified using monoclonal antibodies directed either at the entire T-cell population (OKT3) or the T-cell inducer subset (OKT4). By indirect immunofluorescence, it was shown that the human T-cell population contains no detectable Ia+ cells in the resting state. In contrast, in excess of 60% of the T-cell population expresses Ia antigen after alloactivation in the mixed lymphocyte culture. Moreover, these Ia antigens are expressed within both the OKT4+ and OKT4- subsets. Similarly, phytohemagglutinin and concanavalin A induced approximately 20% of peripheral T cells to express Ia antigen and the expression of these antigens is not restricted to either OKT4 subset. In contrast, only the inducer T-cell population which proliferates maximally to soluble antigen expresses Ia antigens after activation by tetanus toxoid. Thus, the expression of human Ia antigens on unique T-cell subsets depends upon the activation stimuli utilized and ability of the individual subset to respond to a given stimulus. Additional studies indicated that Ia antigens appear on previously Ia- T cells after activation and do not result from clonal expansion of a small subset of Ia+ T cells.

https://rupress.org/jem/article-pdf/150/6/1472/1090619/1472.pdf

Ia DETERMINANTS ON HUMAN T-CELL SUBSETS DEFINED BY MONOCLONAL ANTIBODY Activation Stimuli Required for Expression*

Evidence is presented that the OKT4+ T cell subset in man, defined by a monoclonal hybridoma antibody, provides help for B lymphocyte differentiation in a PWM driven system. Both B cell proliferation and intracytoplasmic immunoglobulin synthesis are facilitated by OKT4+ and not by OKT4- T cells. Given earlier studies demonstrating that OKT4+ T cells were necessary for generation of T cytotoxic cells and the present study that OKT4+ T cells are necessary for the differentiation of B cells, it would appear that the OKT4+ population is the major human T helper (inducer) subset.

Further Characterization of the Human Inducer T Cell Subset Defined by Monoclonal Antibody

A monoclonal antibody directed at a determinant on human peripheral blood T cells was produced and characterized. This hybridoma antibody, termed OKT1, was reactive by indirect immunofluorescence with the entire human peripheral blood T cell population and a subset of human thymocytes. In contrast, OKT1 was unreactive with normal B cells, Null cells, macrophages, and ≥90% of human thymocytes. These findings suggested that OKT1 defines a mature T cell differentiation antigen. In support of this notion was the observation that T cell acute lymphoblastic leukemia cells were nonreactive with OKT1, whereas T cell chronic lymphocytic leukemia cells were reactive. Concomitant functional studies on FACS-separated lymphocytes showed that the T cell proliferative responses to mitogens and soluble and cell surface antigens were contained in the OKT1+ population. Fractionation of peripheral blood T cells into strongly and weakly reactive OKT1+ subgroups uncovered no functional T cell heterogeneity. In addition, when the thymocyte population was separated into OKT1+ and OKT1- subsets, only the OKT1+ thymocytes were MLC responsive. However, unlike peripheral T cells, neither the OKT1+ or OKT1- thymocytes proliferated to the mitogens PHA and Con A. Thus, OKT1+ thymocytes are functionally distinct from OKT1+ peripheral blood T cells. These studies show that a hybridoma antibody can be produced that detects a human differentiation antigen that appears during late intrathymic T cell ontogeny and persists on peripheral T cells.

Gideon Goldstein

Papers

A monoclonal antibody reactive with human peripheral blood monocytes.

A monoclonal antibody reactive with the human cytotoxic/suppressor T cell subset previously defined by a heteroantiserum termed TH2.

Ia DETERMINANTS ON HUMAN T-CELL SUBSETS DEFINED BY MONOCLONAL ANTIBODY Activation Stimuli Required for Expression*

Further Characterization of the Human Inducer T Cell Subset Defined by Monoclonal Antibody

A Monoclonal Antibody with Selective Reactivity with Functionally Mature Human Thymocytes and All Peripheral Human T Cells