Alloantigen-specific cytotoxic and suppressor T lymphocytes are derived from phenotypically distinct precursors

Although Leu-2+ (OKT8+) T cells activated in the mixed lymphocyte reaction (MLR) mediate both alloantigen-specific cytotoxicity and suppression of alloantigen-induced proliferation, it is not known whether these functions derive from a single cell type or phenotypically distinct cells. This study was undertaken to examine the alloantigen-specific cytolytic and suppressor potential of two subpopulations of Leu-2+ cells distinguishable from one another on the basis of their binding to the monoclonal antibody 9.3. Leu-2+, 9.3+ and Leu-2+, 9.3- populations were purified from peripheral blood, cultured for 7 days with autologous helper/inducer (Leu-3+) cells and allogeneic non-T cells, and reisolated before testing for cytotoxicity and suppression. All detectable alloantigen-specific cytolytic activity was confined to the Leu-2+, 9.3+ subpopulation. Killing by this subset was specific for the HLA-A and B (class I) major histocompatibility complex (MHC) antigens of the priming cell. By contrast, suppression of proliferation was mediated predominantly by the Leu-2+, 9.3- cells, and suppression by this subpopulation was specific for the HLA-DR (class II) MHC antigens of the priming cell. The development of suppression by Leu-2+, 9.3- cells was unaffected by cyclosporin A (CsA), an agent shown previously to block the development of cytolytic but not suppressor cells in MLR. Alloactivated Leu-2+, 9.3+ cells were slightly inhibitory of fresh MLR, but this effect as well as the development of cytolytic cells was completely abrogated by CsA. These results indicate that suppressor and cytolytic Leu-2+ T cells activated in MLR are derived from distinct precursors separable by antibody 9.3.     

Soluble antigen-primed inducer T cells activate antigen-specific suppressor T cells in the absence of antigen-pulsed accessory cells: phenotypic definition of suppressor-inducer and suppressor-effector cells

This study was undertaken to characterize interactions among human T cell subpopulations involved in the generation of suppressor T cells specific for a soluble antigen. Purified PPD-primed Leu-3+ cells, when co-cultured for 7 days with fresh autologous Leu-2+ cells, induced differentiation of Leu-2+ but not Leu-3+ cells into specific suppressor T cells, which subsequently inhibited the proliferative response of fresh Leu-3+ cells to PPD but not to tetanus toxoid or allogeneic non-T cells. The PPD-specific suppressor effect of activated Leu-2+ cells was not due to altered kinetics of the PPD response and also extended to the secondary response of PPD-primed Leu-3+ cells. Furthermore, only those Leu-2+ cells that lacked the 9.3 marker, an antigen present on the majority of T cells including the precursors of cytotoxic T cells, differentiated into suppressor T cells. To analyze the inducer population, fresh Leu-3+ cells were separated into Leu-3+,8- and Leu-3+,8+ subpopulations with anti-Leu-8 monoclonal antibody, activated with PPD, and then were examined for inducer function. Although both Leu-3+,8- and Leu-3+,8+ cells proliferated in response to PPD and upon activation expressed comparable amounts of HLA-DR (Ia) antigens, the Leu-3+,8+ subpopulation alone induced Leu-2+ cells to become suppressor-effectors in the absence of PPD-pulsed autologous non-T cells. Once activated, however, Leu-2+ suppressor cells inhibited the PPD response of both Leu-3+,8- and Leu-3+,8+ cells. These results indicate that antigen-primed Leu-3+,8+ inducer cells can directly activate Leu-2+, 9.3- precursors of antigen-specific suppressor T cells in the absence of antigen-pulsed autologous non-T cells.     

Production of B cell growth factor by a Leu-7+, OKM1+ non-T cell with the features of large granular lymphocytes (LGL)

The present study reports the characterization of a non-T cell from human peripheral blood which is capable of releasing BCGF. This BCGF-producing non-T cell had a T3-, T8-, Leu-7+, OKM1+, HLA-DR-, Leu-11- surface phenotype and was likely to belong to the so-called large granular lymphocyte (LGL) subset because: after fractionation of non-T cells according to the expression of Leu-7 or HLA-DR markers, it was found in the Leu-7+, HLA-DR- fractions that were particularly enriched in LGL; it co-purified with LGL on Percoll density gradients; and it expressed Leu-7 and OKM1 markers that are shared by a large fraction of LGL. Although co-purified with cells with potent NK capacities, the BCGF-producing cell was not cytotoxic, because treatment of Leu-7+ cells with Leu-11 monoclonal antibody and complement abolished the NK activity but left the BCGF activity unaltered. The factor released by this LGL subset was not IL 1 or IL 2 mistakenly interpreted as BCGF, because: a) cell supernatants particularly rich in BCGF activity contained very little or no IL 1 or IL 2; b) BCGF-induced B cell proliferation was not inhibitable by anti-Tac antibodies (this in spite of the expression of IL 2 receptor by a proportion of activated B cells); and c) BCGF activity was absorbed by B but not T blasts.     

Interleukin 2-activated human lymphocytes exhibit enhanced adhesion to normal vascular endothelial cells and cause their lysis

When cultured with native or recombinant interleukin 2 (IL 2), human lymphoid cells proliferate and acquire the ability to lyse both NK-sensitive and NK-resistant tumor targets. Such IL 2-activated killer (IAK) cells generally do not destroy nonmalignant nontransformed cells. Due to their apparent specificity for tumor cells, adoptive immunotherapeutic trials of IAK cells and IL 2 have been initiated, with promising results. However, infusion of high doses of IL 2 causes systemic toxicity in patients and experimental animals resulting in the development of a vascular leakage syndrome. Certain aspects of such toxicity suggest IL 2-induced, cell-mediated destruction of normal tissue. This study examines the interaction between IL 2-induced human lymphoid cells and endothelial cells (EC). IL 2, in a dose-dependent manner, causes lymphocytes to strongly adhere to EC, but not to tumor cells, fibroblasts, or epithelial cells. In addition, these IL 2-activated lymphocytes were highly cytotoxic not only to NK-resistant Daudi cells but also to vascular and corneal EC. The IAK cells caused lysis of not only human EC but also bovine EC. Although IAK cells did not display significant adherence to normal human fibroblasts or epithelial cells, when brought together by 50 X G centrifugation, these targets were lysed by IAK cells. The ability to lyse EC was not confined to any single subpopulation of IL 2-activated lymphocytes. The lysis of EC was mediated by both IL 2-activated large granular lymphocytes and small agranular lymphocytes. Furthermore, cells within both CD4+ and CD8+ sublineages of T cells, and also non-T subpopulations, mediated IL 2-induced cytolysis of EC. The destruction of EC by IAK cells may contribute in part to the systemic toxicity associated with infusions of high doses of IL 2.     

Predominance of helper-inducer T cells in mesenteric lymph nodes and intestinal lamina propria of normal nonhuman primates

To define the characteristics of T cells associated with the gastrointestinal tract, the phenotypes and immunoregulatory function of T cells from mesenteric lymph node (MLN) and lamina propria lymphocytes (LPL) were compared to peripheral blood (PBL) and spleen lymphocytes in normal nonhuman primates. Mesenteric lymph node lymphocytes were characterized by a higher proportion of Leu-3+(CD4+) and 9.3+(alpha-Tp44) lymphocytes and a lower proportion of Leu-2+(CD8) lymphocytes than lymphocytes in other sites. LPL and MLN lymphocytes were both characterized by a higher proportion of cells having the helper-inducer phenotypes (Leu-3+, Leu-8+, Leu-3+, 2H4+) compared to PBL. A lower proportion of cells with the suppressor-inducer phenotypes (Leu-3+, Leu-8+, Leu-3+, 2H4+) was found in LPL, but not in MLN lymphocytes compared to PBL. In studies of the Leu-2+ T cells, it was found that whereas PBL, spleen, and LPL contained approximately equal proportions of Leu-2+, Leu-15+ (suppressor phenotype) and Leu-2+, 9.3+ lymphocytes (cytolytic T-cell phenotype), the MLN T cells were predominantly Leu-2+, 9.3+. Furthermore, the Leu-3/Leu-2 ratio was significantly higher in MLN compared to other sites. In pokeweed mitogen-stimulated cultures, the highest helper function for Ig synthesis was found in MLN. Cells from none of the sites studied showed evidence of increased suppressor cell activity. These results show that MLN and LPL T cells in normal nonhuman primates differ from T cells in peripheral blood and spleen. While both MLN and LPL have a high proportion of T cells with the helper-inducer phenotype, cells with the suppressor-effector phenotype are infrequent in MLN, while cells with the suppressor-inducer phenotype are infrequent in LPL.     

Human lymphokine-activated killer (LAK) cells: identification of two types of effector cells

We analyzed the antigenic phenotype of lymphokine-activated killer (LAK) effector cells. Human blood lymphocytes were cultured for 3 days with 100 U/ml recombinant interleukin 2 (rIL 2), subpopulations isolated with monoclonal antibodies and a fluorescence-activated cell sorter (FACS) and assayed for cytotoxic activity against 51chromium labeled noncultured melanoma tumor cells. Initial experiments compared the LAK effector function of CD5+ T lymphocytes vs CD5- cells (predominantly CD16+ NK cells). The mean percent specific release at a 10:1 effector:target (E:T) ratio was 25% +/- 16 for CD5- cells, 10% +/- 6 for CD5+ cells, and 22% +/- 9 for unsorted cells. In contrast, when lymphocyte subpopulations were isolated before rIL 2 culture (LAK precursors), CD5- cells but not CD5+ cells developed LAK activity (28% +/- 12 vs 1% +/- 1, mean percent specific release, 10:1 E:T ratio), confirming our previous results showing that only CD16+ cells were LAK precursors. The discrepancy between LAK effector and precursor phenotypes suggested that LAK precursors acquired CD5 determinants during rIL 2 culture; however, double label immunofluorescence of rIL 2 cultured CD16+ cells showed that this was not the case. The data suggested that in the presence of other cell types, some T lymphocytes may develop LAK activity, but purified blood T lymphocytes do not develop LAK function when cultured with rIL 2 alone. We also analyzed LAK effector function in lymphocyte subpopulations defined by CD4 and CD8 antigens. The data showed that lymphocytes with a low density expression of CD8 and no expression of CD4 were enriched for LAK effector cells, whereas CD4+ and CD8- had less activity than unsorted cells. Lymphocytes with a high density expression of CD8 had activity similar to unsorted cells. We also assessed the contribution of Leu-7 (HNK-1) granular lymphocytes to LAK effector function. After culture with IL 2, lymphocytes were depleted of Leu-7+ cells by antibody and complement treatment and then were sorted into CD5+ and CD5- fractions. The cytotoxic activity of Leu-7-CD5+ cells was a mean 5% +/- 5 vs a mean 14% +/- 8 for the total CD5+ population (20:1 E:T ratio). The activity of Leu-7- CD5- was slightly less than the total CD5- fraction (21% +/- 9 vs 28% +/- 14, 10:1 E:T ratio). In conclusion, LAK effector function was highest in non-T cell (CD5- CD16+) populations and some activity was also present in T cell populations (CD5+ and predominantly Leu-7+).     

A human alloreactive inducer T cell clone that selectively activates antigen-specific suppressor T cells

We showed previously that T cells with the phenotype Leu-3+,8+ are required for the induction of antigen-specific Leu-2+ suppressor cells. Furthermore, when mixed lymphocyte reactions are carried out in the presence of 1 microgram/ml cyclosporin A (CsA), such cultures lead preferentially to the activation of alloantigen-specific suppressor-inducer Leu-3+,8+ cells. In an attempt to generate a clone of T cells with such specific suppressor-inducer properties, we activated Leu-3+,8+ T cells with allogeneic (HLA-DR4+) lymphocytes in the presence of CsA. Clone SP-21, derived by propagating such activated T cells with conditioned medium containing IL 2, is a noncytotoxic, nonsuppressor clone that specifically proliferates to allogeneic cells bearing HLA-DR4 antigen. When cultured with fresh autologous Leu-2+ cells in the absence of HLA-DR4+ cells, clone SP-21 selectively activates Leu-2+ suppressor cells, which inhibit the response of fresh Leu-3+ cells to DR4+ stimulator cells. On the other hand, clone SP-21 fails to induce cytolytic T cells or to help B cell differentiation. These results demonstrate that a T cell clone with a remarkably narrow functional repertoire nonetheless contains and transmits all of the signals necessary for the activation of antigen-specific suppressor cells.     

In situ identification of cells in human leprosy granulomas with monoclonal antibodies to interleukin 2 and its receptor

Leprosy is a chronic granulomatous disease with an immunologic spectrum in which lepromatous leprosy patients have defective cell-mediated immune responses, in comparison to tuberculoid leprosy patients. Immunoregulatory aspects of this spectrum were investigated by using monoclonal antibodies to interleukin 2 (IL 2), IL 2 receptors (Tac), and T lymphocyte subpopulations with immunoperoxidase techniques on frozen sections of skin biopsy specimens from 10 tuberculoid and 10 lepromatous patients. A comparison of IL 2+ cells revealed markedly fewer IL 2+ cells in lepromatous specimens (lep. 0.028% +/- 0.02 vs tub. 0.46% +/- 0.28, p less than 0.001). These IL 2+ cells were large, exhibited cytoplasmic staining, and on double immunostaining were Leu-4+, Leu-3a+, Leu-2a-, Tac-, and OKT6-, consistent with the fact they are IL 2 producers. Equivalent numbers of Tac+ cells were observed in both lepromatous and tuberculoid granulomas (lep. 1.5% +/- 0.5 vs tub. 2.1% +/- 0.7, p, NS), suggesting that the responder cells are present in both conditions. The tuberculoid granuloma was highly organized, composed of a central core of mature macrophages, Leu-3a+ and Tac+ cells with a surrounding mantle of Leu-2a+, Leu-3a+, IL 2+, Tac+, and OKT6+ cells. In lepromatous granulomas, Leu-2a+, Leu-3a+, Tac+, and rare IL 2+ cells were randomly admixed with bacilli-laden macrophages. The defective cell-mediated immune responses in lepromatous leprosy appears to be associated with diminished IL 2 production and disorganization of the granuloma.     

Functional characterization of human T lymphocyte subsets distinguished by monoclonal anti-leu-8

Previous studies have shown that monoclonal anti-Leu-8 antibody identifies functionally distinct subpopulations within both the Leu-2 (T8+) and Leu-3 (T4+) lineages of human T lymphocytes. We now report in detail on the tissue distribution of the Leu-8 antigen and on extensive functional studies of T cells subsets distinguished by their expression or lack of expression of this marker. Leu-8 is present on a wide variety of hematologic cells, including granulocytes, T and B lymphocytes, monocytes, and null or NK cells. Within lymph nodes and tonsils, Leu-8 is absent from both B and T cells within germinal centers but is present on nearly all paracortical lymphocytes. Leu-8 is present on most but not all EBV-transformed B cell lines, reflecting its presence on a subset of normal peripheral blood B cells. None of six malignant T cell lines tested were Leu-8+, whereas most circulating T cells are Leu-8+. Although standard immunoprecipitation techniques failed to demonstrate any specific bands on SDS polyacrylamide gels, the antigenic determinant recognized by anti-Leu-8 is protein or protein-associated, because brief treatment of target cells with pronase abrogated binding of anti-Leu-8. Both Leu-3+8+ and Leu-3+8- cells proliferated in response to several soluble antigens and to autologous and allogeneic non-T cells. Nonetheless, nearly all of the helper T cells for PWM- and AMLR-induced PFC were contained within the Leu3+8- subset. Optimal suppression of the PWM-induced PFC response required both Leu-2+8+ and Leu-2+8- cells, and irradiation of either subset with 3000 R abrogated the capacity of the recombined subsets to effect suppression. In contrast to help for B cell differentiation, both Leu-3+8+ and Leu-3+8- cells were capable of amplifying the development of allospecific T killer cells; precursor and effector T killer cells could be found within both Leu-2+8+ and Leu-2+8- subpopulations. The correlation between Leu-8 phenotype and selected immune functions of T cells (and B cells; see companion paper) indicates that anti-Leu-8 distinguishes important immunoregulatory T and B lymphocyte subsets in man.     

Immunoregulatory T cell circuits in man. Identification of a distinct T cell subpopulation of the helper/inducer lineage that amplifies the development of alloantigen-specific suppressor T cells

Regulation of the immune response in man is dependent on interactions between cells of helper/inducer (Leu-3+/T4+) lineage and cells of suppressor/cytotoxic (Leu-2+/T8+) lineage. By using the mixed leukocyte reaction (MLR) as a model system, we have shown previously that alloantigen-primed Leu-3+ cells induce autologous Leu-2+ cells to differentiate into suppressor T cells that specifically inhibit the response of fresh T cells to the original allogeneic stimulator cells. The current study was undertaken to analyze the roles in this suppressor circuit of subpopulations of Leu-3+ cells distinguished from one another on the basis of their binding or lack of binding to monoclonal anti-Leu-8 antibody. Although both Leu-3+,8- and Leu-3+,8+ T cells proliferated in allogeneic MLR, alloactivated Leu-3+,8+ cells alone induced proliferation and differentiation of Leu-2+ suppressor cells. Leu-3+,8+ cells also induced Leu-3+,8- cells to proliferate, and following their activation in this manner, such autoactivated Leu-3+,8- cells augmented the differentiation of Leu-2+ suppressor cells, but only in the presence of alloactivated Leu-3+,8+ cells. Furthermore, this effect, like the suppressor effect, was specific for the inducer cells, and thus indirectly for the HLA-DR antigens of the original allogeneic stimulator cells as well. These results indicate that alloantigen-primed Leu-3+,8+ cells not only activate specific Leu-2+ suppressor cells but also activate specific Leu-3+,8- suppressor-amplifier cells, and in combination, these cells exert potent feedback inhibition of MLR.     

Differential expression of ecto-5' nucleotidase activity by functionally and phenotypically distinct subpopulations of human Leu-2+/T8+ lymphocytes

Subsets of Leu-2+/T8+ cytotoxic/suppressor T lymphocytes were isolated by using various methods of purification and were investigated for expression of ecto-5' nucleotidase (5'NT) enzyme activity by radiochemical, cytochemical, and ultrastructural techniques. By using both the radiochemical and the cytochemical methods. T4-OKM1- cells displayed higher 5'NT activity in comparison with the entire T4- subpopulation. Analyses of the subpopulations of T4- (and predominantly Leu-2+) cells defined by the Leu-15 or Lyt-1 (9.3) monoclonal antibodies demonstrated that T4-Leu-15- and T4-Lyt-1+ cells displayed high 5'NT activity, whereas virtually no activity was present in T4-Leu-15+ and T4-Lyt-1-cells. At the ultrastructural level, the 5'NT reaction product was detected on the plasma membrane of a proportion of nongranular Leu-2+/T8+ lymphocytes, but no activity was found on cells with a granular lymphocyte (GL) morphology. 5'NT activity was also analyzed in peripheral blood mononuclear cells from one patient with expanded numbers of GL and two patients with GL leukemia. The enzymatic activity was significantly lower in these patients than in normal controls. This study provides new cytochemical evidence demonstrating the heterogeneity of Leu-2+/T8+ cells, and indicates that the population with the suppressor phenotype and function (Leu-15+/Lyt-1-, GL morphology) displays low or absent 5'NT activity, whereas the population composed of cytotoxic cell precursors (Leu-15-/Lyt-1+, nongranular morphology) has high 5'NT activity. Implications of these data for the interpretation of low 5'NT activity described in several immunodeficiency states and lymphoproliferative disorders are discussed.     

Two-color flow cytometry and functional analysis of lymphocytes cultured from human renal allografts: identification of a Leu-2+3+ subpopulation

The phenotype of T lymphocyte subsets present in renal biopsies showing acute cellular allograft rejection in six patients on cyclosporine have been characterized in situ by immunoperoxidase staining, and after expansion in vitro in interleukin 2 (IL-2) by two-color flow cytometry, sorting, and functional analysis. After 8 to 42 days in organ culture, both Leu-3+ (CD4) and Leu-2+ (CD8) subsets were found in each culture, in a ratio that varied from 0.2 to 5.0, which was not significantly different than the results of in situ immunoperoxidase staining of the uncultured biopsy. The cultured cells were almost all Leu-4+ (CD3) T cells (89% +/- 4), which expressed the activation markers DR (82% +/- 6) and the IL 2 (CD25) receptor (15% +/- 4). The Leu-3+ cells were largely Leu-8- (90% +/- 6), whereas a minority of the Leu-2+ cells were Leu-15+ (CD11) (26% +/- 4). Only a small fraction of the Leu-2+ cells stained for Leu-7 (8% +/- 6). Functional analysis of FACS-purified Leu-2-3+ and Leu-2+3- populations indicated that both subsets proliferated in response to graft donor antigens in a mixed lymphocyte reaction (MLR) and produced IL 2. Only the Leu-2+3- population demonstrated donor-specific cytotoxic activity. A minor subpopulation in each culture were both Leu-3+ and Leu-2+ (2.0%). Leu-2+3+ cells from one biopsy were purified to homogeneity (99.8%), and were found to express the T cell antigen receptor complex Ti/CD3 (WT-31+, Leu-4+), but not the common thymocyte antigen CD1 (OKT6). The Leu-2+3+ cells neither responded in the MLR, nor showed any cytotoxic capacity. The Leu-2+3+ cells were capable of IL 2 but not interferon-gamma production. None of the purified cultures demonstrated NK activity. A subset of the purified Leu-2+3+ cells lost Leu-2+ during 1 to 3 wk in culture, and became Leu-2-3+. These studies provide evidence that the cells that infiltrate renal allografts during rejection include alloproliferative, lymphokine-producing cells of both Leu-2+ and Leu-3+ subsets. The Leu-2+3- cells are also highly cytotoxic against donor lymphocytes, indicating the presence of helper independent cytotoxic T cells. A minor population of Leu-2+3+ T cells that do not express donor specific function was also identified.     

In vivo effects of recombinant IL-2. I. Isolation of circulating Leu-19+ lymphokine-activated killer effector cells from cancer patients receiving recombinant IL-2

This study was designed to isolate and phenotypically characterize lymphokine-activated killer (LAK) cells generated in vivo during administration of high dose rIL-2 to cancer patients. The development of circulating LAK effector cells in these patients was demonstrated by the ability of fresh PBL to exhibit lytic activity against the NK-resistant Daudi cell line and fresh tumor cells without prior in vitro culture with rIL-2. Kinetic studies demonstrated that circulating LAK effector cells are detectable 4 to 6 wk after the initiation of rIL-2 therapy. Cells isolated by FACS revealed that circulating LAK cells are Leu-19+, Leu-17+ but CD5-. We have previously reported that circulating Leu-19+ cells are heterogeneous with regard to the expression of CD16 and CD8. Since sorting of cells expressing Leu-19 and either low quantities of CD8 or CD16 resulted in cytolytic activity in both the positive and negative fractions, these latter two markers do not identify subpopulations of Leu-19+ cells with or without LAK cytolytic activity. Although all LAK cells generated in vivo were Leu-19+, we generated LAK cells from the Leu-19- subpopulation after in vitro culture with rIL-2, suggesting that at least some of in vitro generated LAK cells are derived from Leu-19- precursor cells. These LAK cells did not, however, express the Leu-19 surface marker. Based on the functional data reported in this paper, we conclude that circulating LAK effector cells are a phenotypically heterogeneous population that express surface Ag in association with NK cells and not T lymphocytes.     

Characterization of interleukin 2-expanded human peripheral blood lymphocytes: not only NKH-1+-NK cells but also NKH-1+ and NKH-1- T cells are LAK effectors

In vitro culture of human peripheral blood mononuclear cells (PBMC) with interleukin 2 (IL-2) results in the expansion of lymphocytes including lymphokine-activated killer (LAK) cells. Using flow cytometry, studies were undertaken to determine the phenotype and LAK activity of each subset of lymphocytes expanded in vitro as a result of incubation for 2 weeks with 2500 U/ml of recombinant IL-2. Such expanded PBMC, when examined by two-color staining with various combinations of anti-CD3, 4, 8, 16, and NKH-1 monoclonal antibodies, consisted of the following six subgroups of cells: (1) CD3+4+8-, (2) CD3+4-8+, (3) CD3+4-8-, (4) CD3-16+NKH-1+, (5) CD3-16-NKH-1+, and (6) CD3-16-NKH-1-. Of the six subgroups, all five subgroups that could be tested, i.e., CD3+ T cells (CD3+4+8-, CD3+4-8+, CD3+4-8-), CD16+ natural killer (NK) cells (CD3-16+NKH-1+), and CD3-16-NKH-1- non-T non-NK cells, possessed LAK activity. Both NKH-1- as well as NKH-1+ T and non-T cells possessed LAK activity.     

CD4+ Leu-8+ T cell supernatant activity that inhibits Ig production.

The subpopulation of CD4+ T cells that expresses the Leu-8 peripheral lymph node homing receptor suppresses PWM-stimulated Ig synthesis. To determine the mechanism of this suppression, the immunoregulatory activity of culture supernatants obtained from peripheral blood CD4+ Leu-8+ T cells cultured with anti-CD3 mAb and PMA (Leu-8+ supernatant) was determined. Leu-8+ supernatant suppressed PWM-stimulated Ig synthesis in cultures containing non-T cells and CD4+ Leu-8- T cells. In contrast, the supernatant from CD4+ Leu-8- T cells did not suppress Ig synthesis. The inhibitory activity of CD4+ Leu-8+ T cell supernatants could not be accounted for by a deficiency or excess of IL-2, IL-4, IFN-gamma, IL-6, or PGE2. In studies examining the effect of CD4+ Leu-8+ supernatant on T cells, the supernatant did not alter either mitogen-induced proliferation or the helper function of CD4+ Leu-8- T cells. In studies examining the effect of CD4+ Leu-8+ supernatant on B cells, the supernatant inhibited Staphylococcus aureus Cowan I strain-induced B cell Ig secretion but not B cell proliferation. The suppressor activity of Leu-8+ supernatant was eliminated by protease treatment and was eluted by HPLC in two main peaks, with molecular sizes of 44 and 12 kDa. In summary, these studies indicate that supernatants from activated CD4+ Leu-8+ T cells directly suppress B cell Ig production.     

Lectin-dependent and anti-CD3 induced cytotoxicity are preferentially mediated by peripheral blood cytotoxic T lymphocytes expressing Leu-7 antigen

Monoclonal antibodies against the CD3 antigen and certain lectins can induce interleukin 2 dependent antigen-specific T cell clones to mediate non-antigen specific cytotoxicity. On the basis of this observation, we predicted that it may be possible to identify cytotoxic T lymphocytes (CTL) in peripheral blood without knowing the antigen specificity of these in vivo primed CTL. By using this strategy, peripheral blood lymphocytes were separated into low and high-density fractions on Percoll gradients and were tested for cytotoxic activity in the presence or absence of concanavalin A (Con A) or anti-Leu-4 antibody. Lectin-dependent cellular cytotoxicity (LDCC) and anti-CD3 induced cytotoxicity against both natural killer (NK)-insensitive and NK-sensitive targets were exclusively mediated by low-density CD3+ T lymphocytes. Additional studies indicated that low-density CD3+ T lymphocytes co-expressing Leu-7 antigen preferentially mediated this activity, although in some individuals, significant activity was also observed in the low-density T cells lacking Leu-7. In contrast, high-density CD3+ T lymphocytes and CD16+ (Leu-11+) NK cells (both Leu-7 and Leu-7+) did not mediate nonantigen-specific cytotoxicity under these conditions. The finding that NK cell-mediated cytotoxicity was unaffected by these lectins refutes the hypothesis that lectin-dependent cellular cytotoxicity is simply a result of effector and target agglutination. T cell-mediated cytotoxicity was both lectin and antibody specific. Phytohemagglutinin, Con A, and pokeweed mitogen induced cytolytic activity in the Leu-7+ T cells, whereas wheat germ agglutinin did not. Of the antibodies against T cell-associated differentiation antigens (anti-Leu-2,3,4, and 5), only anti-Leu-4 induced cytotoxicity. This anti-CD3-induced cytotoxicity was essentially completely inhibited by the presence of anti-LFA-1 or anti-CD2 monoclonal antibodies, implicating these molecules in the triggering process. A proportion of the CD3+, Leu-7+ CTL expressed HLA-DR antigens, indicating possible in vivo activation. Because previous clinical studies have indicated that lymphocytes with this phenotype may be elevated in clinical situations associated with immunosuppression and chronic viral infection, this unique subset of CD3+ T lymphocytes may represent a population of in vivo primed CTL possibly against viral antigens.     

Phenotype of chronic lymphocytic leukemia (CLL) B-cells. B-CLL cells express the Leu-8 antigen

In the present report we studied the phenotype of peripheral blood mononuclear cells (PBMC) from 25 patients with B-cell chronic lymphocytic leukemia (CLL). Cells from all the cases expressed monoclonal surface immunoglobulins (SmIg), formed rosettes with mouse erythrocytes (MRFC) and were positive with OKB 2 and OKIa monoclonal antibodies. In addition, CCB 1 monoclonal antibody was positive in 17 out of 20, Leu-1 in 18 out of 21 and Leu-8 in 23 out of 25 cases. Double labelling experiments confirmed that the Leu-8 antigen was co-expressed on Leu-1+, CCB2+, HLA-DR+ B-CLL cells. Thus, B-CLL cells generally express the SmIg+, MRFC+, Leu-1+, OKB2+, Leu-8+ phenotype. Since it is known that normal peripheral blood B cells may be divided into two subpopulations according to Leu-8 expression, our data indicate that B-CLL cells originate from the more immature Leu-8+ B-cell subset which will respond to anti-IgM, whereas it reacts poorly to pokeweed mitogen.     

Identification of a cord blood T cell subset of CD3+4-8-45R+ suppressing interleukin 2 production in the autologous mixed lymphocyte reaction and the mode of action of exogenous IL2 in the induction of IL2 production

As previously reported, the inability of cord blood T cells to produce IL2 in the autologous mixed lymphocyte reaction (AMLR) could be recovered by the treatment of stimulator non-T cells with interferon-gamma (IFN-gamma) and of the AMLR with exogenous IL2. In the present study, we showed that addition of untreated autologous cord blood T cells to the above-mentioned AMLR abrogated the IL2 production in a dose-dependent manner, suggesting active suppression by the untreated T cells because untreated cord blood T cells did not consume IL2. Suppressor activity was abrogated by the treatment of cord blood T cells with monoclonal anti-CD3 antibody plus complement or with monoclonal anti-CD45R (Leu 18) antibody, but not by the treatment with monoclonal anti-CD4 antibody and/or anti-CD8 antibody plus complement. These data showed that the cord blood suppressor T cells were CD3+4-8-45R+. This suppressor activity also disappeared by culturing with rIL2 for 8 hr. As the frequency of CD45R+ cord blood T cells was comparable to that of CD45R+ adult T cells and was minimally affected by the IL2 treatment, functional modulation of CD45R+ suppressor T cells by IL2 is suggested. Moreover, in spite of the inhibitory effect of anti-CD45R antibody on the suppressor activity, IL2 production was not induced merely by addition of anti-CD45R antibody directly to the responder cells in AMLR. Taken together, these data suggest the requirement of exogenous IL2 for IL2 production in that IL2-producing-precursor T cells themselves should be stimulated by IL2 in addition to the modulation of CD45R+ suppressor T cells by IL2.     

Antigenic, functional, and molecular genetic studies of human natural killer cells and cytotoxic T lymphocytes not restricted by the major histocompatibility complex

Cytotoxicity not restricted by the major histocompatibility complex (MHC) is mediated by two distinct types of lymphocyte: natural killer (NK) cells and non-MHC-restricted cytotoxic T lymphocytes (CTL). These two types of cytotoxic lymphocytes can be distinguished by antigenic phenotype, function, and molecular genetic studies. In human peripheral blood, NK cells are identified by expression of the Leu-19 and/or CD16 cell surface antigens, and lack of CD3/T cell antigen receptor (Ti) complex expression (i.e., CD3-,Leu-19+). Peripheral blood non-MHC-restricted CTL express both CD3 and Leu-19 (i.e., CD3+, Leu-19+, referred to as Leu-19+ T cells). Both Leu-19+ T cells and NK cells lyse "NK-sensitive" hematopoietic tumor cell targets, such as K562, without deliberate immunization of the host. However, most "NK activity" in peripheral blood is mediated by NK cells, because they are usually more abundant and more efficient cytotoxic effectors than Leu-19+ T cells. The cytolytic activity of both NK cells and Leu-19+ T cells against hematopoietic targets was enhanced by recombinant interleukin 2 (rIL 2). NK cells, but not peripheral blood Leu-19+ T cells, were also capable of lysing solid tumor cell targets after short-term culture in rIL 2. Southern blot analysis of NK cells revealed that both the T cell antigen receptor beta-chain genes and the T cell-associated gamma genes were not rearranged, but were in germ-line configuration. These findings indicate that NK cells are distinct in lineage from T lymphocytes and do not use the T cell antigen receptor genes for target recognition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bacterial activation of human natural killer cells. Characteristics of the activation process and identification of the effector cell

We showed previously that contact of human peripheral blood lymphocytes with glutaraldehyde-fixed Salmonella bacteria augmented their cytotoxic capacity against NK-sensitive targets. We have now analyzed the characteristics of the activation and also identified the subsets of lymphocytes responding to bacterial contact. Blocking of protein synthesis with cyclohexamide totally abrogated bacterial induction of activated killing (AK), whereas inhibition of DNA synthesis with mitomycin C did not significantly affect the capacity of lymphocytes to respond to bacterial contact. Both the induction and the effector phase of AK were radioresistant. The AK cells exhibited efficient lytic activity, comparable to that induced by recombinant IL 2 (rIL 2), against NK-resistant targets (including both hematopoietic and solid tumor cell lines). All inducible cytotoxic activity was contained within the subset of lymphocytes expressing Leu-19 (NKH-1) antigen. Leu-19- lymphocytes exhibited no significant NK activity and could not be further stimulated by bacterial contact, rIL 2, or IFN-alpha. Within the Leu-19+ lymphocyte subset, two distinct cell types were present; CD3-, Leu-19+ NK cells and CD3+. Leu-19+ T cells. The CD3+, Leu-19+, T cells mediated low levels of non-MHC-restricted cytotoxicity against K562, but did not respond to bacterial contact, even though rIL 2 could augment their lytic activity slightly. However, the cytotoxic activity of CD3-, Leu-19+ NK cells was significantly augmented by bacterial contact. Within the CD3-, Leu-19+ NK cell population both CD16+ and CD16- cells responded to bacterial activation. The CD3-, CD16-, Leu-19+ cells constituted 1 to 4% of the Percoll-fractionated low buoyant density lymphocytes and accounted for the activation seen within the CD16- lymphocyte population. Thus bacterial stimulation of NK activity seems to be mediated for the most part via CD16+, Leu-19+ cells, and a minor overall contribution is mediated via CD3-, CD16-, Leu-19+ cells. No apparent involvement of T cells was seen in the lytic response of lymphocytes to bacterial contact.