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.     

Immune status of Greek patients with beta-thalassemia major negative for anti-HIV

Patients with thalassemia who receive multiple blood transfusions are at risk for the acquired immunodeficiency syndrome. Peripheral blood lymphocyte subpopulations were studied in 22 multitransfused thalassemic patients; 10 patients were without splenectomy and 12 were studied after splenectomy. Both groups were negative for anti-HIV. Four additional patients who were found positive for anti-HIV and ten healthy controls were also included in this study. Patients without splenectomy compared to controls and to patients after splenectomy showed a significant decrease of both percentage (p less than 0.001) and absolute numbers (p less than 0.001) of Leu-7+ cells without significant abnormalities of T4/T8 ratio (1.56 +/- 0.4). Patients after splenectomy compared to controls and to patients without splenectomy showed a significant increase of the absolute numbers of lymphocytes and lymphocytes subsets T11+, T3+, T4+, T8+ and SmIg+ cells. In the seropositive patients for HIV only a significant increase of the absolute number of T8+ cells was observed while the T4/T8 ratio was 1.24 +/- 0.73. The decrease in the percentage of Leu-7+ cells in patients without splenectomy correlated inversely to the total amount of blood transfused. In conclusion patients with thalassemia had normal T4/T8 ratio and did not show the abnormal immunologic profile that has been reported in haemophiliacs.     

Distribution of peripheral blood leukocytes in acute heatstroke.

We examined 11 heatstroke patients (mean rectal temperature 41.4 +/- 0.3 degrees C) and 40 healthy subjects to determine the effects of hyperthermia on peripheral blood leukocyte distribution. Precooling samples were taken on admission. Whole blood was incubated with conjugated monoclonal antibodies, and erythrocytes were eliminated by FACS lysing solution. Lymphocyte subsets were detected by specific mouse monoclonal antibodies: Leu-4/CD3+ (T-cells), Leu-3a/CD4+ (T-helper cells), Leu-2a/CD8+ (T-suppressor-cytotoxic cells), Leu-11/19/CD16+/CD56+ (natural killer cells), and Leu-12/CD19+ (B-cells). Immunofluorescence was measured with a flow cytometer. The number of circulating leukocytes and lymphocytes was significantly increased in heatstroke patients. This lymphocytosis was mainly due to an increase in T-suppressor-cytotoxic cells and natural killer cells. The absolute number of lymphocytes and T-suppressor-cytotoxic cells significantly correlated with the degree of hyperthermia (r = 0.62, P = 0.04; r = 0.751, P = 0.007, respectively). There was a significant decrease in the percentages of T-, B-, and T-helper cells and increase in T-suppressor-cytotoxic and natural killer cells, giving a marked decrease in the ratio of T-helper to T-suppressor-cytotoxic cells. We conclude that heatstroke is associated with leukocytosis and significant alteration in absolute number and percentage of circulating lymphocyte subpopulations.     

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.     

Two-color immunofluorescence and flow cytometry analysis of lymphocytes in long-term renal allotransplant recipients: identification of a major Leu-7+/Leu-3+ subpopulation

Using two-color immunofluorescence with fluorescein isothiocyanate (FITC)- and phycoerythrin (PE)-labeled monoclonal antibodies to human lymphocyte antigens and flow cytometry, we studied lymphocyte subsets in 16 long-term renal allotransplant recipients at risk for a mean of 78 +/- 15 mo. The absolute number of Leu-1+, Leu-2a+, and Leu-3a+ lymphocytes is significantly decreased compared with a control population, whereas Leu-7+ and Leu-15+ subsets remain unchanged despite standard chronic immunosuppression (azathioprine and prednisone). Within the Leu-7+ subset, we found various phenotypes. Doubly fluorescent lymphocytes Leu-7+/Leu-1+ and Leu-7+/Leu-2a+ are not significantly different in the transplant population compared with a normal control population. The Leu-7+/Leu-3a+ subpopulation is seen to be significantly elevated, and the Leu-7+/Leu-15+ subpopulation decreases significantly. The relationship between the modification of these two phenotypes within the Leu-7 subset may be an important correlate of decreased NK cell activity in long-term renal allotransplant recipients. These Leu-7+/Leu-3a+ cells, normally less than 1% of peripheral blood lymphocytes, have no known functional activity.     

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.     

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.     

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.     

Effect of infectious factors on human and animal cytogenetic structures]

The analysis of blood leucocyte chromosomes has been carried out on 60 patients with different infectious diseases (influenza, measles, scarlet fever, and disentery), and on 47 patients immunized against measles, tick-born encephalitis, typhoid fever and brucellosis. The mutagenic influence of viruses on the genital cells of mice and on the human somatic cells in vitro was studied. Both viruses and bacteria appeared to be able to bring about different breaks in human and animal cells.     

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.     

The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes

We examined the antigenic and functional characteristics of human peripheral blood lymphocytes that differentially express the CD16 (Leu-11) and Leu-19 (NKH-1) antigens. Leu-19 is a approximately 220,000 daltons protein expressed on approximately 15% of freshly isolated peripheral blood lymphocytes. Within the Leu-19+ subset, three distinct populations were identified: CD3-,CD16+,Leu-19+ cells; CD3+,CD16-,Leu-19+ cells; and CD3-,CD16-,Leu-19bright+ cells. Both the CD3+,CD16-,Leu-19+ and CD3-,CD16+,Leu-19+ populations mediated non-major histocompatibility complex (MHC)-restricted cytotoxicity against the NK-sensitive tumor cell K562 and were large granular lymphocytes. CD3-,CD16+,Leu-19+ NK cells were the most abundant (comprising approximately 10% of peripheral blood lymphocytes) and the most efficient cytotoxic effectors. The finding that CD3+,Leu 19+ lymphocytes mediated cytotoxicity against K562 unequivocally demonstrates that a unique subset of non-MHC-restricted cytotoxic CD3+ T lymphocytes are present in the peripheral blood of unprimed, normal individuals. However, CD3+,CD16-,Leu-19+ cells comprised less than 5% of peripheral blood lymphocytes, and the cytotoxic activity of this subset was significantly less than CD3-,CD16+,Leu-19+ NK cells. Most CD3+,Leu-19+ T cells co-expressed the CD2, CD8, and CD5 differentiation antigens. The antigenic and functional phenotype of peripheral blood CD3+,Leu-19+ cytotoxic T lymphocytes corresponds to the interleukin 2-dependent CD3+ cell lines that mediate non-MHC-restricted cytotoxicity against NK-sensitive tumor cell targets. A small population of Leu-19bright+ lymphocytes lacking both CD3 and CD16 was also observed. This population (comprising less than 2% of peripheral blood lymphocytes) contained both large agranular lymphocytes and large granular lymphocytes. CD3-,CD16-,Leu-19bright+ lymphocytes also mediate non-MHC-restricted cytotoxicity. The relationship of these CD3-CD16-,Leu-19bright+ lymphocytes to CD3+ T cells or CD16+ NK cells is unknown.     

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)     

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.     

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.     

T lymphocyte cytotoxicity with natural varicella-zoster virus infection and after immunization with live attenuated varicella vaccine

Varicella-zoster virus (VZV) specific cytotoxicity was investigated during acute primary VZV infection, in naturally immune subjects and after vaccination with the live attenuated varicella vaccine by using T cell cultures (TCC) generated by stimulating PBMC with VZV Ag and autologous VZV-superinfected lymphoblastoid cell lines as targets. Lysis of VZV-infected lymphoblastoid cell lines was observed by TCC from acutely infected subjects, naturally immune subjects, and recipients of the varicella vaccine. VZV glycoprotein I induced cytotoxic T cells but killing was less efficient than killing by TCC stimulated with VZV Ag. The TCC were primarily CD4+ (mean 86.6%) T lymphocytes with 15.2% of the cells coexpressing Leu-19. TCC were predominantly restricted by HLA class II as demonstrated by lack of any blocking using class I mAb and blocking of 15 to 71% by L243, a mAb to class II. Unrestricted killing as measured by killing of K562 cells occurred in all TCC but was minimally greater than that observed against uninfected autologous targets. Phenotypes of PBMC during acute infection had an initial increase in CD4+ cells and an overall decrease in the percentage of circulating Leu-11+ (CD16). No enhanced K562 killing was demonstrated in PBMC from subjects with acute infection compared to subjects without infection. CD4+ CTL may function as an important primary host response in acute varicella. Immunization with live attenuated varicella vaccine induced VZV-specific, memory CTL responses comparable to those of naturally immune subjects. The demonstration of their persistence long after primary VZV infection may indicate a role for CTL in restriction of viral replication during episodes of VZV reactivation from latency.     

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.     

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.     

Colony formation by subpopulations of human T lymphocytes. III. Antigenic phenotype and function of colony-forming cells, colony cells, and their expanded progeny

The antigenic phenotype of individual PHA-induced T lymphocyte colonies was studied with a direct immunofluorescence technique using fluorescein-labeled anti-Leu-2a and anti-Leu-3a antibodies. Of the colonies grown from mononuclear peripheral blood cells 85% were Leu-3a+ (inducer/helper phenotype), 12% were Leu-2a+ (suppressor/cytotoxic phenotype), and 3% contained equal numbers of Leu-2a+ and Leu-3a+ cells. Fluorescence-activated cell sorter (FACS) separated T-cell subsets showed that Leu-2a+ cells and Leu-3a+ cells form exclusively Leu-2a+ and Leu-3a+ colonies, respectively. Leu-3a+ cells formed colonies in both the absence and presence of conditioned medium (PHA-CM), whereas colony formation by Leu-2a+ cells was absolutely dependent on PHA-CM. Mixing experiments with FACS-separated T-cell subsets showed that Leu-2a+ cells inhibit colony formation by Leu-3a+ cells in a cell dose-dependent manner both in the presence and absence of PHA-CM. Phenotype analysis of individual colonies from mixing experiments strongly suggested monoclonal proliferation in the present colony assay system. The majority of expanded T-cell colonies showed helper activity in a reverse hemolytic plaque-forming B-cell assay, although to a lesser degree as compared to that of freshly isolated T lymphocytes.     

Cell numbers and in vitro responses of leucocytes and lymphocyte subpopulations following maximal exercise and interval training sessions of different intensities.

In vitro lymphocyte function and the mobilisation of peripheral blood leucocytes was examined in eight trained subjects who undertook an incremental exercise test to exhaustion and a series of interval training sessions. Venous blood samples were obtained before the incremental test, immediately after, and 30, 60, and 120 min after the test. Interval training sessions were undertaken on separate days and the exercise intensities for each of the different sessions were 30%, 60%, 90% and 120% of their maximal work capacity respectively, as determined from the incremental exercise test. There were 15 exercise periods of 1-min duration separated by recovery intervals of 2 min in each session. Venous blood samples were obtained immediately after each training session. Significant increases in lymphocyte subpopulations (CD3+, CD4+, CD8+, CD20+, and CD56+) occurred following both maximal and supramaximal exercise. This was accompanied by a significant decrease in the response of cultures of peripheral blood lymphocytes to Concanavalin A (ConA), a T-cell mitogen. The state of lymphocyte activation in vivo as measured by CD25+ surface antigen was not, however, affected by acute exercise. The total number of lymphocytes, distribution of lymphocyte subpopulations and in vitro lymphocyte response to ConA had returned to pre-exercise levels within half an hour of termination of exercise but serum cortisol concentrations had not begun to fall at this time. There was a significant decrease in the CD4+:CD8+ cell ratio following exercise; this was more the result of increases in CD3-CD8+ cells (CD8+ natural killer cells) than to CD3+CD8+ cells (CD8+ T-lymphocytes). Decreased responsiveness of T-cells to T-cell mitogens, postexercise, may have been the result of decreases in the percentage of T-cells in postexercise mixed lymphocyte cultures rather than depressed cell function. The cause of this was an increase in the percentage of natural killer cells which did not respond to the T-cell mitogen. The results indicated that while a substantial immediate in vitro "immunomodulation" occurred with acute exercise, this did not reflect an immunosuppression but was rather the result of changes in the proportions of reactive cells in mononuclear cell cultures. We have also demonstrated that the degree of the change in distribution of lymphocyte subpopulation numbers and responsiveness of peripheral blood mononuclear cells in in vitro mitogen reactions increased with increasing exercise intensity. Plasma volume changes may have contributed to some of the changes seen in leucocyte population and subpopulation numbers during and following exercise.     

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.