Lymphocyte subpopulations in the neonate: identification of an immature subset of OKT8-positive, OKT3-negative cells

T cell subpopulations of lymphocytes from cord blood (CBL) of 24 newborns and from peripheral blood (a-PBL) of 24 healthy adult volunteers were assessed in T cell-enriched, T cell depleted and unseparated lymphocyte fractions by using OKT3, 4, 6, and 8 monoclonal antibodies. The results show that T cell-enriched CBL include adult numbers of OKT3+, OKT4+, OKT6+ and OKT8+ lymphocytes whereas the T cell-depleted fraction consists of a high percentage of OKT8+, OKT3-, non-E rosette-forming cells bearing a PNA receptor. The presence of the PNA receptor and the lack of the OKT3+ antigen strongly support the hypothesis that the subset of OKT8+ cells in cord blood includes immature T lymphocytes that may represent an intermediate stage between thymocytes and mature peripheral T cells.     

Spontaneous human T-cell cytotoxicity against murine hybridomas expressing the OKT3 monoclonal antibody: comparison with natural killer cell activity

Human peripheral blood lymphocytes (PBL) exhibited spontaneous cytotoxicity against OKT3 monoclonal antibody (mAb)-expressing murine hybridoma cells (OKT3 hybridomas). In contrast, other murine hybridomas expressing OKT4, OKT8, anti-HLA DR, and anti-HLA A, B, and C mAb were not lysed. PBL showed much lower levels of cytotoxicity (3 folds) against OKT3 hybridomas as compared with NK activity against the K562 targets. Lymph node (LN) cells exhibited the inverse relationship of cytotoxicity levels. The addition of OKT3 mAb to the effector cells totally blocked both the binding and the lysis of OKT3 hybridoma targets, indicating that the CD3 antigen on the effector cells may be involved in recognition of the targets. The addition of concanavalin (Con A) also inhibited the cytotoxicity of OKT3 hybridomas. OKT4 mAb-expressing hybridomas became susceptible to lysis after chemical attachment of OKT3 mAb with CrCl3. The kinetics of lysis of OKT3 hybridomas resembled that of NK activity. Both cytotoxicities were detectable after 1 to 2 hr and reached plateau levels by 4 to 6 hr. Effector cells responsible for lysis of OKT3 hybridomas expressed T3, T8, and Leu 7 antigens, but lacked T4 and Leu 11b antigens, and were sensitive to the treatment with L-leucine methyl ester. These results indicate that T3+, T8+, Leu 7+ and T4-, and Leu 11- granular lymphocytes have a spontaneous cytotoxic activity against OKT3 hybridomas which is different from classic NK activity. These findings may provide a method for the assessment of T-cell cytotoxicity mediated presumably by in vivo generated cytotoxic T lymphocytes in blood and the other immune organs.     

Ly1+ PRO-B lymphocyte clones. Phenotype, growth requirements and differentiation in vitro and in vivo.

Several clones obtained from the bone marrow of a BALB/c mouse were found to contain the heavy and light chain Ig genes in the germline configuration, to express Ly1 and to carry the B cell lineage markers B-220, Lyb8 and BP-1; these clones are Pgp-1+, LFA-1+, J11d+, Mac-1+ and Thy1-, Lyt2-, L3T4-, GM1.2- and Ia-. Three clones analyzed in detail (Lyd9, LyH7 and Lyb9) have receptors for interleukin (IL) 2 and IL3 as assessed with the 7D4 and CC11 monoclonal antibodies respectively. They grow in rIL3 but not in rIL2 or rIL1; both rIL4 and rIL5 also promote their proliferation, albeit to a much lesser extent than rIL3. None of the interleukins tested alone or in various combinations promoted the clones to differentiate in vitro along the B cell pathway. Treatment with 5-Azacytidine (5-Aza) induced cell surface Ia expression but not rearrangement or expression of Ig genes. However, 5-Aza-treated Lyd9, LyH7 and Lyb9 cells co-cultured with X-ray irradiated accessory cells and LPS gave rise to Ly1+, IgM+ B lymphocytes (range 14-51%) including mu + kappa + (78-93%), and mu + lambda + (9-25%) B lymphocytes. In vivo, the Lyd9, LyH7 and Lyb9 clones gave rise to IgM+ B lymphocytes (8.5-17%) including mu + kappa +, and mu + lambda +, but not to Lyt2+ or L3T4+ T lymphocytes after 4-6 weeks of transfer into Scid mice. Our results indicate that Ly1+ IgM+ cells comprise a subpopulation of B lymphocytes that is derived from IL3-responsive Ly1+ PRO-B lymphocytes.     

Immunobeads test (antibody coated polyacrylamide beads) in the immunological characterization of lymphoproliferative disorders

Three groups of patients with immunoproliferative disorders (15 multiple myeloma, 11 non-Hodgkin's lymphoma, 21 chronic lymphocytic leukemia) were studied by immunological characterization and compared to a group of 20 normal subjects (controls) using anti-immunoglobulin coated polyacrylamide beads (T-B Quantigen test, QT), erythrocyte rosettes (ER), surface immunoglobulin (SIg), and monoclonal antibodies for T and B cells (OKT3; OKT11; OKT8; OKT4; IaDR); null cells (NC) and double marker (DM) cells were also considered. The values for normal subjects for T-B, NC and DM cells were comparable. Results for the patient groups strikingly differed. There were progressively larger differences between the T and B percentages obtained with different techniques. The largest differences were seen in patients with chronic lymphocytic leukemia and the smallest in multiple myeloma patients; values were intermediate in non-Hodgkin lymphoma. The different findings were related to the number of DM cells (ER+, SIg+ QT+) and the different tests used. The importance of these findings in the diagnostic approach to lymphoproliferative disorders is discussed.     

Production of alpha-lymphotoxin by human T-cell subsets

Human T cells were isolated from peripheral blood lymphocytes (PBL) and sensitized to allogeneic PBL in a one-way mixed-lymphocyte culture. These sensitized T cells were fractionated on the basis of their possession of Fc receptors for IgG (TG+) or IgM (TM+), or the absence of both IgG and IgM receptors (TG-M-). When restimulated with alloantigen of the same derivation, TG+, TM+, and TG-M- cells yielded almost equal amounts of cytotoxin. Anti-alpha-lymphotoxin serum neutralized most of this cytotoxic activity indicating that alpha-lymphotoxin (alpha-LT) constituted most of this activity. Although TG-M- cells function as effectors in allogeneic cytotoxicity, TG+ cells lyse IgG-coated targets in an antibody-dependent cell-mediated cytotoxic (ADCC) reaction, which has been shown to be mediated in part by alpha-LT. Whether TM+ cells can be cytotoxic is not clear. In addition, freshly isolated human T-cell subsets were stimulated with phytohemagglutinin-P (PHA-P). After PHA stimulation, TG+, TM+, and TG-M- cells produced similar amounts of soluble cytotoxin, which was largely neutralized by anti-alpha-LT. The TG+ cells incorporated less thymidine than the TM+ or TG-M- cells. Likewise, OKT4+ and OKT8+ subsets, isolated with the aid of monoclonal OKT8 or OKT4 antibody and complement, yielded lymphotoxin after stimulation with PHA. It is shown that all T-cell subsets, as defined here, can produce lymphotoxin. Furthermore, depending on the assay system, cytotoxicity can be clearly demonstrated in all of these subsets, except in TM+ cells, where positive and negative results have been reported.     

Characterization of human large granular lymphocyte subpopulations: comparison of the phenotype of NK cells and of interleukin 2-dependent progenitors of cytolytic effector cells

Antigenically different subpopulations of human large granular lymphocytes (LGL) were identified according to their reactivity with monoclonal antibodies (MoAb). Antigen-positive and -negative subsets were isolated by immunoaffinity columns using a Sepharose 4B gel coupled with F(a')2 goat anti-mouse IgG or by flow cytometry cell sorting. The distinct LGL subsets were tested for natural killer (NK) activity against a panel of tumor targets: K562, Daudi, Alab; and for antibody-dependent cellular cytotoxicity (ADCC) against antibody-coated RL male 1 cells. LGL positively selected for any of the following phenotypic markers: B73.1+, OKM1+, OKT11+, and OKT10+ were highly cytotoxic, while B73.1- and OKM1- cells were completely devoid of NK activity. The OKT10- and OKT11- LGL subsets were occasionally cytotoxic, with low levels of reactivity. LGL subpopulations were also tested in a limiting dilution assay (LDA) for their capacity to proliferate in medium supplemented with interleukin 2 (IL-2) and to develop NK-like cytotoxic activity. The majority of proliferative progenitors have the following phenotype: OKT11+, OKM1-, B73.1-, and OKT10-, while the majority of progenitors for cytotoxic cells were OKT11+, OKM1+/-, OKT10+, and B73.1-. Results indicate that although B73.1+ cells can grow, the mature B73.1+ NK cells seem to be primarily derived in vitro from a small subset of less differentiated B73.1 pre-NK progenitors in the peripheral blood lymphocytes.     

Studies on human blood lymphocytes with iC3b (type 3) complement receptors. I. Granular, Fc-IgG receptor positive and negative subsets in healthy subjects and patients with systemic lupus erythematosus

By using the OKM1 monoclonal antibody and the fluorescence-activated cell sorter to identify lymphocytes bearing iC3b (type 3) complement receptors, two principal populations of OKM1+ lymphocytes have been identified in human peripheral blood. One subset exhibited azurophilic granules and Fc receptors for IgG stained by Leu-11. The other population did not display FcR, but was enriched in cells reacting with OKT3 and OKT8 (low intensity). In healthy subjects, approximately 60% of CR3+ lymphocytes were granular FcR-bearing cells and only 18% co-expressed OKT3 determinants. In patients with systemic lupus erythematosus (SLE), CR3+ lymphocytes were predominantly FcR negative cells and 71% lacked granules. Only 33% reacted with Leu-11, but 50% co-expressed OKT3, 44% reacted with OKT8+, and 15% were OKT4+. We tested the hypothesis that agranular OKT3+ Leu-11- lymphocytes, such as those found in SLE patients, contained the precursors of natural killer (NK) cells. Leu-11+ cells were removed from normal lymphocytes by complement lysis, and the remaining cells were treated with recombinant IFN-alpha, IFN-gamma, or IL 2. These procedures were ineffective in generating typical NK effector cells. Our studies do not support the hypothesis that CR3+ Leu-11- lymphocytes are the precursors of granular Leu-11+ NK cells.     

Characterization of recirculating lymphocytes in rheumatoid arthritis patients: selective deficiency of natural killer cells in thoracic duct lymph

Five patients with rheumatoid arthritis (RA), who were treated by lymphocyte depletion by using thoracic duct drainage (TDD), provided an opportunity to characterize the phenotype and function of their recirculating lymphocytes. We found that: a) thoracic duct lymphocytes (TDL) were similar in their proportion of T cells (83% +/- 6 OKT3+), OKT4+ subset (65% +/- 8), and OKT8+ subset (22% +/- 6) to peripheral blood lymphocytes (PBL): b) fewer natural killer-like cells were present in TDL (5% +/- 4 Leu-7+; 2% +/- 2 Leu-11+: 8% +/- 2 OKM -1+) than in PBL (20% +/- 10 Leu-7+: 11% +/- 6 Leu-11+; 18% +/- 5 OKM -1) (p less than 0.01); c) TDL differed from synovial fluid lymphocytes ( SFL ) and synovial membrane lymphocytes ( SML ) in that TDL lacked a high percentage of activated lymphocytes (T cells bearing Ia antigen, OKT10 , and transferrin receptor): d) immature T cells (expressing either OKT6 antigen or reactive with peanut agglutinin) were not found in TDL even late in the course of TDD: and e) in vitro functional studies demonstrated that TDL were similar to PBL in their ability to synthesize immunoglobulin after mitogen stimulation and to generate cytotoxic T lymphocytes capable of lysing autologous EBV-transformed B cells. However, natural killer activity, as measured by lysis of K562 cells was significantly lower in TDL than PBL (p less than 0.05). These results demonstrate that natural killer cells defined by phenotype and function are excluded from thoracic duct lymph and thus have a circulation pattern different from most T cells.     

Interleukin 2 production by human T lymphocytes identified by antibodies to dipeptidyl peptidase IV

T-Cell subsets identified by polyclonal and monoclonal antibodies to dipeptidyl peptidase IV (DP IV) were investigated. Analysis in a cytofluorograf revealed 63 +/- 7% positive scatter-gated T lymphocytes. DP IV-positive cells were found to be T11+, 74-81% OKT4+, and 12-19% OKT8+. DP IV-negative cells were T11+ and comprise 16-40% OKT8+, and 10-30% OKT4+ T cells. Treatment of T lymphocytes with rabbit anti-DP IV and complement as well as the presence of rabbit anti-DP IV during culture resulted in a reduction of interleukin 2 (IL-2) production. This reduction was not observed with the mouse monoclonal anti-DP IV antibody II-19-4-7. Positive enrichment of DP IV-positive lymphocytes by cell sorting revealed excellent IL-2 production of DP IV-positive cells and very poor IL-2 activity in supernatants obtained from DP IV-negative lymphocytes. Thus, DP IV may serve as cell surface marker for IL-2-producing T lymphocytes.     

Chemotaxis of large granular lymphocytes

The hypothesis that large granular lymphocytes (LGL) are capable of directed locomotion (chemotaxis) was tested. A population of LGL isolated from discontinuous Percoll gradients migrated along concentration gradients of N-formyl-methionyl-leucyl-phenylalanine (f-MLP), casein, and C5a, well known chemoattractants for polymorphonuclear leukocytes and monocytes, as well as interferon-beta and colony-stimulating factor. Interleukin 2, tuftsin, platelet-derived growth factor, and fibronectin were inactive. Migratory responses were greater in Percoll fractions with the highest lytic activity and HNK-1+ cells. The chemotactic response to f-MLP, casein, and C5a was always greater when the chemoattractant was present in greater concentration in the lower compartment of the Boyden chamber. Optimum chemotaxis was observed after a 1 hr incubation that made use of 12 micron nitrocellulose filters. LGL exhibited a high degree of nondirected locomotion when allowed to migrate for longer periods (greater than 2 hr), and when cultured in vitro for 24 to 72 hr in the presence or absence of IL 2 containing phytohemagluttinin-conditioned medium. The chemotactic LGL was HNK-1+, OKT11+ or HNK-1+, OKT11- on the basis of monoclonal antibody and complement depletion. They did not bear either T cell or monocyte cell surface markers, exhibiting an OKT3-, OKT4-, OKT8-, OKM1-, and MO2- phenotype, and did not form E rosettes at 29 degrees C, which is characteristic of lytic NK cells in contrast to T cells. Furthermore, a rat LGL leukemia (RNK) exhibited a chemotactic response to both f-MLP and casein. LGL chemotaxis to f-MLP could be inhibited in a dose-dependent manner by the inactive structural analog CBZ-phe-met, and the RNK tumor line specifically bound f-ML[3H]P, suggesting that LGL bear receptors for the chemotactic peptide.     

OKT8 antibody inhibits OKT3-induced IL 2 production and proliferation in OKT8+ cells

We studied IL 2 production and proliferation induced by OKT3 mitogenic monoclonal antibody in the OKT8+ T cell subset. OKT3 antibody induced IL 2 production and proliferation in OKT8+ cells in a typical time-dependent manner: maximal IL 2 levels were found in 24 hr culture supernatants; maximal proliferation was found on day 3. OKT3 antibody was mitogenic over a wide range of concentrations (0.125 to 500 ng/ml). The presence of OKT8 antibody (greater than or equal to 100 ng/ml) in these cultures resulted in almost complete inhibition of IL 2 production and proliferation. Kinetic studies demonstrate that OKT8 antibody suppresses both IL 2 production and response to exogenous IL 2 in OKT8+ cells when added within the first 2 hr of culture. After 14 to 20 hr of culture, addition of OKT8 only blocks IL 2 production but not the IL 2 response of activated OKT8+ cells. The specificity of inhibition by OKT8 antibody of OKT3 mitogenicity on OKT8+ cells was confirmed by the failure of Leu-I and OKT4 antibody to produce the same effect and by the lack of inhibition by OKT8 antibody of OKT3-induced IL 2 production and proliferation in OKT4+ cells.     

The recognition specificity of a murine helper T cell for hemagglutinin of influenza virus A/PR/8/34

Human large granular lymphocytes (LGL), which are known to be responsible for natural killer (NK) cell activity, also produced a variety of lymphokines including interleukin 2 (IL 2), colony stimulating factor (CSF), and interferon (IFN) in response to phytohemagglutinin (PHA) or concanavalin A (Con A). Human peripheral blood LGL, which were purified by removal of monocytes adhering to plastic flasks and nylon columns, followed by separation on a discontinuous Percoll gradient, and additional treatment with anti-OKT3 and Leu-M1 plus complement, were more potent producers of these lymphokines than unseparated mononuclear cells (MNC), nylon column-eluted cells, or purified T lymphocytes. Moreover, IL 2 production by LGL could be further distinguished in that it was not enhanced by the addition of macrophages or macrophage-derived factor, i.e., IL 1, whereas addition of macrophages did potentiate IL 2 production by T lymphocytes. Further analysis of cells in the LGL population using various monoclonal antibodies revealed that removal of cells with OKT11 or AF-10, a monoclonal antibody against human HLA-DR antigen, decreased IL 2 production, whereas removal of OKT8+, OKM1+, Leu-M1+, or Leu-7+ cells led to enhanced IL 2 production. The LGL population is therefore heterogeneous and includes at least three functionally and phenotypically distinct subsets. An atypical T cell subset (OKT3-, Leu-1-, OKT11+) rather than the myeloid subset of LGL (Leu-M1+ or OKMI+) was the source of LGL-derived IL 2, whereas the latter subset and/or another subset of OKT8+ cells appear to regulate this IL 2 production. In addition to performing NK activity, LGL on a per cell basis seem to be more effective than T lymphocytes in producing lymphokines, namely, IL2, CSF, and IFN.     

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.     

Circadian and seasonal changes of the inducer:suppressor ratio (OKT4+:OKT8+) in venous blood of healthy adults

Circadian and seasonal variations in the T helper: T suppressor-cytotoxic ratio were investigated in peripheral blood from five healthy young men. Mononuclear cells were isolated on Ficoll-Paque gradient, then incubated with OKT4 and OKT8 monoclonal antibodies. Plasma cortisol was determined in four of these seven time series. Large interindividual differences were documented and statistically validated for the 24-hr.-means of total lymphocytes, OKT4+:OKT8+ ratio, and of plasma cortisol (both total and free). For a pooled data, a circadian rhythm was demonstrated by cosinor (p less than 0.001) for total lymphocytes (acrophase at 1.00 hr.), total plasma cortisol (acrophase at 10.30 hrs.) and free plasma cortisol (acrophase at 9.50 hrs.), but not for OKT4+:OKT8+ ratio. This index however exhibited a statistically significant circadian rhythm in April and August, but not in November. Its double-amplitude exceeded 80% of the 24-hour-mean and its acrophase was localized at 6.40 hrs. in April and at 22.30 hrs. in August. Its 24-hr-mean was higher in August as compared to April and November. The circadian rhythm in the OKT4+:OKT8+ ratio did not seem to be related to that of plasma cortisol. Both circadian and seasonal variations need to be taken into account when investigating the regulations of immune variables such as T helper: T suppressor-cytotoxic ratio.     

Preliminary characterization of accessory cells in the nonadherent fraction of human blood mononuclear cells

We investigated why peripheral blood mononuclear cells rigorously depleted of adherent cells by sequential incubation on plastic and nylon wool remained fully responsive to both antigenic and mitogenic signals. Nylon wool nonadherent cells (NWNA) depleted of cells expressing HLA-DR by monoclonal antibody and complement lysis did not respond to tetanus toxoid (TT) or suboptimal concentrations of phytohemagglutinin (PHA). Addition of adherent accessory cells to these NWNA HLA-DR- cells reconstituted the response to stimuli. NWNA, fractionated by discontinuous density gradient centrifugation, contained high density cells which were unresponsive alone to optimal concentrations of both TT and PHA. All the lower density fractions contained cells which were accessory for higher density cell responses to stimuli. The lowest density fraction was approximately 30% monocytes (esterase and peroxidase positive) and less than or equal to 3% B lymphocytes (surface IgG bearing). The other low density fractions contained large granular lymphocytes but rarely monocytes and no B lymphocytes. Depletion of OKT3+, OKM1+, and Leu-11+ cells from lower density cells by monoclonal antibody and complement lysis did not abolish their accessory activity, but depletion of HLA-DR+ cells or gamma irradiation of these cells decreased their accessory activity for PHA and eradicated accessory activity for TT. Thus, the responsiveness of NWNA to soluble antigenic and mitogenic signals is due, in part, to the presence of low density cells which are radiosensitive and phenotypically HLA-DR+ OKT3-OKM1-Leu-11-. Accessory activity in NWNA seems to reside, therefore, in a cell which is not a typical monocyte, natural killer cell, nor B or T lymphocyte.     

Modulation of T lymphocyte differentiation antigens: influence of aging

In vitro modulation of human T lymphocyte surface differentiation antigens T3, T8, and T4, by their respective monoclonal antibodies, was studied as a function of donor age. Kinetic studies performed on lymphocytes from young adults indicated that modulation is dependent on concentration of antibody used and duration of culture. OKT3 modulates T3 rapidly (maximum at less than 24 hr) and relatively completely (79% at the highest concentration of antibody used). By 48 hr, regeneration of the T3 antigen is apparent. T8 modulation by OKT8 is slower (continued modulation at 48 hr) and less complete than is T3 modulation by OKT3. OKT4 does not modulate the T4 antigen. In elderly individuals modulation of T3 by OKT3 is preserved whereas modulation of T8 by OKT8 is significantly reduced (24 +/- 8% at 48 hr vs 53 +/- 4% for young controls). These observations document further age-related changes in properties of human T suppressor cells.     

Differential recovery of circulating T cell subsets after nodal irradiation for Hodgkin's disease

To better understand the immunologic effects of lymphoid irradiation (LI), blood levels of T cell subsets were sequentially monitored in 15 patients before, during, and after irradiation treatment for Hodgkin's disease. Blood levels of all lymphocytes, T cells, and T cell subsets (defined by OKT4 and OKT8) fell dramatically and in similar proportions during early therapy, reaching levels less than 20 to 25% of control by the completion of mantle irradiation, and continuing at very depressed levels through the completion of therapy. Blood levels of OKT8-reactive (OKT8+) cells returned to pretreatment levels (402 +/- 38/mm3 vs 360 +/- 32/mm3 pretreatment) between 6 to 8 mo after LI, whereas blood levels of OKT4-reactive (OKT4+) cells returned to only 42% of previous values (242 +/- 22/mm3 vs 584 +/- 34/mm3 pretreatment) over the same period. The pre-LI ratio of OKT4+ to OKT8+ cells was 1.85 +/- 0.24 and fell to 0.65 +/- 0.05 between 6 to 8 mo after LI. During the recovery period, discrepancies of 208 +/- 32 cells/mm3 (3 to 5 months post LI) and 198 +/- 32 cells/mm3 (6 to 8 mo post LI) developed between the blood levels of OKT3+ cells and the sum of OKT4+ and OKT8+ cells. This suggests the emergence of OKT4+/OKT3-, OKT8+/OKT3-, and/or OKT4+/OKT8+ cells. In five patients, the sum of OKT4+ and OKT8+ cells was compared with the number of cells simultaneously co-stained by OKT4 and OKT8. It appeared that a significant proportion of the cells were OKT4+/OKT3- and OKT8+/OKT3- lymphocytes. We concluded that LI is similarly cytotoxic to peripheral blood T cell subpopulations. The reversed ratio after LI is a result of a slower repopulation of the peripheral blood by OKT4+ cells relative to OKT8+ cells. T cells after LI show a high degree of antigenic immaturity. It is postulated that the bone marrow that lies outside the fields of treatment and contains predominantly immature OKT8+/OKT3- cells is a major source of T cells repopulating the peripheral blood after LI.     

Development and distribution of B lineage cells in the domestic cat: analysis with monoclonal antibodies to cat mu-, gamma-, kappa-, and lambda-chains and heterologous anti-alpha antibodies

To trace the development and distribution of B lineage cells in the domestic cat (Felis catus), we have produced monoclonal antibodies against mu-, gamma-, kappa-, and lambda-chains of feline immunoglobulins (Ig). Goat antibodies against human mu-, alpha-, and lambda-chains, which are reactive with shared determinants on their feline counterparts, were used in conjunction with the panel of mouse monoclonal antibodies. Cytoplasmic mu+ pre-B cells and surface IgM+ B lymphocytes were observed in 42 day fetal liver in which pre-B cells were more abundant than IgM+ B cells. Pre-B cells also were found in bone marrow in young cats, and continued to be generated in the marrow throughout life. In the spleen, adult levels of B cells were attained by 12 wk of age, at which time the frequencies of surface IgM+, IgG+, and lambda+ cells were 49, 3, and 40%, respectively. The distributions of Ig isotypes also were determined among plasma cells as a function of age and tissue localization. IgM plasma cells were predominant in the bone marrow of 1-wk-old cats, whereas IgG plasma cells were the prevalent isotype in adult bone marrow. In the mesenteric lymph nodes of adult animals, the frequency distributions of IgM, IgG, and IgA plasma cells were similar to the frequency distributions of IgM, IgG, and IgA isotypes among bone marrow plasma cells. IgA+ plasma cells predominated in the intestinal lamina propria, in which IgG+ and IgM+ plasma cells were relatively infrequent. In the tissues of both young and adult animals, the ratio of lambda:kappa expression was approximately 3:1. We conclude that the pattern of B cell development in the cat resembles that found in other mammals, except that the kappa to lambda ratio is reversed.     

Subsets of human large granular lymphocytes (LGL) exhibit accessory cell functions

The present study shows that human large granular lymphocytes (LGL) depleted of OKT3 (T lymphocytes) and Leu-M1-positive (monocytes) cells exhibit accessory cell function for the T lymphoproliferative responses to the soluble stimulants Staphylococcus protein A (SpA) or Streptolysin O (SLO), as well as to surface antigens in the autologous and allogeneic mixed leukocyte reaction (MLR). Fractionation of LGL into subsets according to their reactivity with alpha OKT11, alpha DR, and alpha OKM1 MoAb led to the identification of the subset(s) of LGL with OKT11+, DR+, OKM1+ phenotype as the antigen-presenting cell (APC), whereas the DR-, OKM1- subset(s) of LGL was completely ineffective. Furthermore, virtually all the natural killer (NK) activity of LGL was associated with OKT11+ and OKM1+, DR+ LGL that exerted the observed APC function, suggesting that NK-active cells may also act as effective APC for T lymphocyte activation. These results indicate that human LGL with NK activity may exert other noncytotoxic functions and may play a major role in immunoregulation.     

Monocyte-independent interleukin-2 production and proliferation of human T cells in response to murine hybridomas expressing the OKT3 monoclonal antibody: interleukin-1 is not required for T-cell proliferation

We report a new, monocyte-independent system for the induction of activation and proliferation of human T cells in response to murine hybridomas expressing the OKT3 monoclonal antibody (OKT3 hybridomas). Incubation of nylon-wool-nonadherent (NA) lymphocytes or purified T cells with OKT3 hybridomas resulted in interleukin-2 (IL-2) production, expression of IL-2 receptor, modulation of the CD3 antigen, and proliferation. In contrast, murine hybridomas (OKT4, OKT8, anti-HLA-DR, and others) expressing monoclonal antibodies (mAb) other than OKT3 did not induce T-cell activation and proliferation. T cells did not respond to OKT3 mAb alone. OKT3 hybridomas alone did not produce interleukin-1 (IL-1) or other soluble factors that might be involved in the induction of IL-2 production by T cells, and they did not contain membrane-bound IL-1. In addition, IL-1 activity was not detected in cultures of NA-lymphocytes and OKT3 hybridomas, clearly demonstrating that IL-1 was not required, at least in this system, for T-cell activation and proliferation. Direct cell-cell contact between T cells and OKT3 hybridomas was required for IL-2 production. Thirty to fifty percent of T cells formed conjugates with the OKT3 hybridomas but not with the OKT4 or OKT8 hybridomas. Both conjugate formation and IL-2 production were significantly inhibited by the OKT3 mAb and by the anti-LFA-1 mAb. The cells responsible for IL-2 production were found to be of the T3+ T4+ T8- Leu 7- Leu 11- phenotype. IL-2 activity produced by NA-lymphocytes in response to OKT3 hybridomas became detectable as early as 1 hr and reached a maximum by 8 hr, preceding IL-2 receptor expression, modulation of the CD3 antigen, and [3H]thymidine incorporation of T cells. T cells produced higher concentrations of IL-2 in response to OKT3 hybridomas than in response to equal numbers of monocytes and OKT3 mAb. Addition of monocytes to cultures of T cells and OKT3 hybridomas resulted in suppression of IL-2 production in a concentration-dependent manner, suggesting that monocytes regulate the levels of IL-2 production. This monocyte-independent system may be useful for further dissection of T-cell activation and proliferation and its regulation by monocytes.