Four distinct antigen systems on human thymus and T cells defined by monoclonal antibodies: immunohistological and immunochemical studies.

Human thymus and T cell antigens were identified by using four distinct monoclonal antibodies (MoAb), designated 2D5, 5B3, 7A5 and 9D4. 2D5 antibody reacted with most human thymocytes and a few peripheral lymphocytes as well as with a subpopulation (20%) of bone marrow cells, and precipitated a 45K molecular weight (mol. wt.) component from 125I-labelled thymus cell lysate. 7A5 antibody also reacted with the majority (80%) of thymocytes but neither with peripheral lymphocytes nor with bone marrow cells. The antigen detected by 7A5 was a glycoprotein consisting of 48K and 12K mol. wt. components, which were non-covalently associated with each other. 5B3 reacted with virtually all of human thymus and T cells but not with the majority of B cells and bone marrow cells. This reagent precipitated a 72K mol. wt. glycoprotein from thymus and T cells. An additional 65K mol. wt. glycoprotein was precipitated by 5B3 together with the 72K mol. wt. component, but with poor reproducibility. 9D4 antibody, on the other hand, reacted with a 200K mol. wt. component from thymus and T cells as well as 220K and 210K components from the non-T cell fraction of tonsil lymphocytes. Whereas antigens detected by 2D5 and 7A5 appeared to be highly expressed on cortical thymocytes, the antigen defined by 5B3 occurred much more abundantly on medullary thymocytes and peripheral T cells than on cortical thymocytes. Based on the data described above, it is suggested that 7A5, 5B3 and 9D4 MoAb recognize human homologues of mouse TL, Ly-1 and Ly-5 antigens, respectively, whereas 2D5 antibody seems to resemble OKT10, as described by others.     

Genetics and strain distribution of concanavalin A-reactive Ly-2-, L3T4- peripheral precursors of autoreactive T cells

Cytotoxic treatment of BALB/c cells from different peripheral lymphoid tissues by a cocktail of monoclonal antibodies against Thy-1, Ly-1, L3T4 and Ly-2 differentiation markers (anti-T cocktail) plus complement eliminates all mature T lymphocytes. Yet a population of dull Thy-1+, Ly-1-, L3T4-, Ly-2-, corresponding to about 1% of the initial population, can be detected by flow cytometry which proliferate under concanavalin A stimulation. These anti-T killing-resistant cells (TKR) were previously shown to be capable of differentiating in culture into class II-restricted autoreactive T helper cells. We demonstrate here that such cells can be detected in mice of BALB/c and DBA/2 genetic background but are absent in C57BL/6 and B10 animals. The presence of TKR cells is dominant in (BALB/c x C57BL/6)F1 hybrids and genetically controlled by two genes which are neither H-2 nor Igh linked. TKR cells are also detected in young NZB mice but disappear with the development of the systemic autoimmune disease in old animals. Thy-1+, L3T4-, Ly-2- cells from MRL lpr/lpr mice also respond to concanavalin A but are removed by the anti-T treatment. Altogether, arguments are presented suggesting that TKR cells represent a particular subset of double-negative peripheral T cells which may correspond to autoreactive T cell recursors that would escape the thymic selection. We postulate that these cells are present in all mouse strains but their susceptibility to killing by anti-Thy-1 antibodies differs depending on background genes.     

Studies of murine lymphocyte alloantigens Ly-7.2

The Ly-7.2 alloantigen has previously been defined by an alloantiserum and shown to be expressed on T and B cells; it is now studied for its distribution on responder and stimulator cells in the mixed lymphocyte reaction, on cells responding to mitogens and on mitogen-induced blast cells. In the mixed lymphocyte reaction, the responder cells were Ly-7.2+; stimulator cells were Ly-7.2-. The Ly-7.2 antigen also had an unusual distribution on cells responding to mitogens: leucoagglutinin-responsive cells were Ly-7.2-, concanavalin A Ly-7.2+, pokeweed mitogen Ly-7.2+/-, and lipopolysaccharide Ly-7.2-, i.e., Ly-7 can distinguish between subpopulations of T cells which respond to mitogens (phytohaemagglutinin and concanavalin A). Ly-7.2 was present on approximately 60% of blast cells induced by all mitogens indicating that both Ly-7.2+ and Ly-7.2- cells could be activated to become Ly-7.2+. Further characterization of concanavalin A and leucoagglutinin-stimulated Ly-7.2+ and Ly-7.2- T blast cells with anti-Ly-2.1 and anti-Ia. 17 monoclonal antibodies demonstrated the presence of several T lymphocyte subsets as both Ly-7+ and Ly-7- blast cells contained Ly-2+, Ly-2-, Ia+ and Ia- cells. Ly-7.2 therefore has a heterogenous distribution on normal and activated T and B cell subpopulations, and is a potentially important antigenic marker for studies of lymphocyte differentiation and function.     

CD4-/CD8-T cells: amplification in spleens of mice following in vivo treatment with monoclonal antibody anti-L3T4

Amplification of L3T4-/Ly-2-(CD 4-/CD 8-) T cells following in vivo administration of anti-L3T4 monoclonal antibody was examined in the spleen of mice by flow cytometry and immunohistochemistry. BALB/c mice were given 4-7 weekly injections of either anti-L3T4 (1 mg/week) or phosphate-buffered saline (control group), and dispersed spleen cells and tissue sections analyzed for the presence of Thy-1.2+, L3T4+, or Ly-2+ cells, and for the absence of both L3T4 and Ly-2 on Thy-1.2+ cells. Prior to treatment, L3T4+ and Ly-2+ cells accounted for virtually all Thy-1.2+ cells in approximately a 2:1 ratio. Following anti-L3T4 treatment, L3T4+ cells were depleted, and Ly-2+ cells accounted for about 2/3 of the Thy-1.2+ cells. A population of L3T4-/Ly-2- T cells was generated that accounted for 20-30% of the Thy-1.2+ cells, accounted for most of the Thy-1.2+ cells in red pulp, and was also present among the predominant Ly-2+ cells in periarteriolar sheaths.     

Generation of L3T4-/Ly-2- T cells in Peyer's patches of mice treated with monoclonal antibody against helper T cells

The effect of in vivo administration of anti-L3T4 monoclonal antibody on the generation of an L3T4-/Ly-2- (CD4-/CD8-) population of Thy-1.2+ cells was examined in Peyer's patches of mice by two-color flow cytometry. Female BALB/c mice aged 8 wk were given 4-6 weekly injections of either anti-L3T4 (1 mg/wk) or phosphate-buffered saline (control group), and dispersed Peyer's patch cells analyzed for the presence and absence of L3T4 and Ly-2 on Thy-1.2+ cells. In anti-L3T4 treated mice, L3T4-/Ly-2- T cells accounted for 25-30% of the Thy-1.2+ cells, whereas in control mice these cells represented only 3-4% of the T cells. The remaining 70-75% of the Thy-1.2+ cells after anti-L3T4 treatment were Ly-2+ and not L3T4+. The L3T4-/Ly-2- population of Thy-1.2+ cells is a novel subset which has not been previously found in Peyer's patches and is amplified when helper T cells are depleted.     

Candida albicans-specific Ly-2+ lymphocytes with cytolytic activity.

To determine whether antigen (Ag)-specific cytotoxic T lymphocytes are generated during experimental Candida albicans infection, purified L3T4+ and Ly-2+ lymphocytes from immunized mice were cultured in the presence of syngeneic accessory cells, C. albicans Ag, and interleukin 2. Yeast-infected bone marrow macrophages were used as target cells in a standard 51Cr-release assay. Freshly isolated L3T4+ and Ly-2+ lymphocytes failed to lyse either target cell type. However, Ag-specific, major histocompatibility complex (MHC)-unrestricted lysis of infected macrophages was evident with immune Ly-2+ cells after 7-10 days in culture. The cultured cells were greater than 98% Thy-1+, CD3+, L3T4-, Ly-2+, T cell receptor alpha/beta + T cells, and their lytic activity was potentiated by the addition of anti-CD3 monoclonal antibodies. At limiting effector cell numbers, Ag-specific MHC-restricted lymphocytes with cytotoxic activity against infected macrophages could be identified. We suggest that C. albicans infection stimulates multiple cytotoxic cell precursors with varying recognition stringency, which include MHC class I-restricted, Ag-specific cytotoxic T lymphocytes.     

Characterization of proximal colonic lymphoid tissue in the mouse

Here we describe a nodule of lymphoid tissue which was consistently located in the proximal colon of mice approximately 25% of the distance from the cecum to the rectum. Immunohistochemical characterization of this nodule demonstrated that the majority of lymphocytes were relatively immature 14.8+ (B220+), IgM+, Ia+ (specificity 20) B cells some of which were also Ly-1+. These nodules also possessed an occasional T cell (Thy-1+, Ly-1+, Lyt-2+) aggregate at the periphery. Rare, small areas did not stain for either T or B cell markers. These lymphoid nodules were associated with epithelial cells which stained positively with the ER-TR4 monoclonal antibody (which also recognizes thymic cortical epithelial cells) and also with ER-TR6, which has been reported to recognize thymic macrophages or dendritic cells. The overlying colonic epithelium stained intensely with the ER-TR4 monoclonal antibody. Proximal colonic lymphoid tissue was extremely sensitive to steroid treatment, losing approximately 80% of its mass within 24 hours in response to a single intraperitoneal injection of 2 mg hydrocortisone acetate. This response was similar to that of the thymus and to that reported for the bursa of Fabricius, but unlike that of other gastrointestinal lymphoid aggregates. These results indicated that proximal colonic lymphoid tissue contains a high frequency of relatively immature B cells and may be a primary site of their generation, possibly including some of the Ly-1+ phenotype. These observations correlate with new evidence suggesting that the allantois participates in the formation of the distal midgut, including its lymphoid components.     

激发型CD28单抗直接激发的T细胞及其表型

1.用羊抗鼠IgG(20μg/ml)包被96孔细胞培养板,再加入游离的激发型CD28单抗(终浓度为5 μg/ml);2.用激发型CD28单抗(10 μg/ml)直接包被96孔细胞培养板,然后分别加入10~5个/孔经E-花结实验获取的人外周血T细胞(PBTC),其中CD3+T>95％。逐日观察细胞的生长状态并绘制生长曲线,用3H-TdR掺入法分析PBTC的增殖效应,用FITC标记的单抗经流式细胞仪(FCM)分析细胞CD3、CD4、CD8、CTLA-4、4-1BB及OX40的表达。逐日细胞计数的结果表明,经羊抗鼠IgG包被后加入游离激发型CD28单抗或直接用激发型CD28单抗包被,均能引发PBTC的活化与增殖效应,激发3 d时的刺激指数(SI)大于9,细胞CD3、CD4、CD8、CTLA-4、4-1BB及OX40的阳性表达率(x±s,％)分别为98.6±0.2、74.4±3.1、22.5±2.0、2.1±0.4、18.4±2.2、35.2±3.5。与XGB7(作为APC)刺激的T细胞相比,经CD28单抗直接激发的T细胞,CD4+/CD8+T细胞的比值及OX40+T细胞的百分率升高(P<0.01),但不表达负性调节分子CTLA-4。     

Monoclonal antibody (WT 1) directed against a T cell surface glycoprotein: characteristics and immunosuppressive activity

WT 1, an IgG2a subclass monoclonal antibody, recognizes a human T lineage specific antigen (mol. wt 40,000). This antigen is strongly expressed on thymic T blasts, and on peripheral T cells activated by phytohaemagglutinin, whereas cortical thymocytes and peripheral T cells are moderately positive for WT 1. In contrast, B lymphocytes, myeloid and erythroid cells, including the progenitor cells of these lineages, and terminal deoxynucleotidyl transferase positive cells in the bone marrow, are all WT 1 negative. Binding of WT 1 to T cells is blocked by a previously described antibody (3A1) suggesting that both antibodies bind to the same antigen present on human T cells. WT 1, however, is also reactive with T lymphocytes from rhesus monkeys whereas 3A1 is not. Therefore, the biological effects of WT 1 could be studied in a monkey model. In a skin allograft model, WT 1 was immunosuppressive and induced a marked prolongation of graft survival.     

Studies on the immunoregulation of thyroid autoantibody production in vitro.

The spontaneous production (without mitogen or antigen) of antithyroglobulin and antimicrosomal antibodies by peripheral (PBL) and thyroid-derived lymphocytes from patients with Hashimoto's thyroiditis (HT) has been studied with particular emphasis on the regulation of this phenomenon. Based on studies of DNA and protein synthesis, kinetic studies and B/T reconstitution experiments, in most HT patients, spontaneous production by PBL is accounted for by secretion of preformed antithyroglobulin (termed Type 1 patients), whereas active production is observed in a small minority (termed Type 2). In none of 24 HT patients could active antimicrosomal antibody production by PBL be detected. Conversely, thyroid-derived lymphocytes produced both autoantibodies by an active process. Pokeweed mitogen (PWM) stimulation enhanced antibody production by PBL in the Type 1 group but not in Type 2 or thyroid-derived lymphocytes. T lymphocytes were required for antibody synthesis in both thyroid antigen-driven and peripheral PWM-driven cultures. By separating T lymphocytes into T4+ (helper) and T8+ (suppressor) subsets with monoclonal antibodies, T-cell modulation of autoantibody production in both systems was studied. In a PWM-induced system, both thyroid and peripheral T-cell subsets were capable of modulating peripheral antibody production. In the thyroid lymphocyte antigen-specific system, further addition of thyroid derived T8+ cells alone caused partial suppression of antibody production but not with peripheral T8+ cells. Of interest was the partial decrease of antibody production by the thyroid lymphocytes by added peripheral T4+ cells. The fact that the production of thyroid autoantibodies by thyroid-derived mononuclear cells (which included T suppressor, T helper and B lymphocytes) could be reduced by the addition of more suppressor T lymphocytes suggests that an antigen-specific defect in the T4+/T8+ thyroid cell balance may account for the in vivo production of these antibodies in patients with Hashimoto's thyroiditis.     

Identification of Ia glycoproteins in rat thymus and purification from rat spleen.

A fraction of la-like glycoproteins was prepared from rat thymocytes by lentil lectin affinity chromatography and gel filtration in deoxycholate. Spleen cells from mice immunized with this preparation were fused with myeloma cells to produce antibody-secreting hybrid cell lines. Antibody from four lines called MRC OX, 3, 4, 5, 6 reacted with the la-like glycoproteins, and MRC OX 3 antibody recognized an antigenie determinant polymorphic in the rat. All four antibodies also bound to mouse spleen cells and all detected polymorphisms. Studies on recombinant mouse strains suggest that the determinants are coded by the I-A subregion of the H-2 complex. MRC OX 3 correlates with Ia specificity 9, while MRC OX 4, 5, 6 correlate with specificity 17 or 18. MRC OX 4 monoclonal antibody was used for affinity chromatography to purify Ia glycoproteins from rat spleen. The rat Ia glycoprotein complex was composed of two noncovalently linked polypeptide chains of apparent mol. wt. (unreduced) 30 000 and 24 000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The purified Ia glycoprotein partially inhibited the binding to thoracic duct lymphocytes of an alloantiserum which detects Ia antigens linked to the major histocompatibility complex. The monoclonal anti-la antibodies bound to the majority of peripheral B lymphocytes and 18% of thymocytes, but did not significantly bind to peripheral T lymphocytes. There were on average 150000 molecules of Ia glycoprotein per la-positive B lymphocyte, and 45 000 molecules per la-positive thymocyte. From the same fusion, another cell line was prepared called MRC OX 2 which secretes monoclonal antibody to a previously undefined thymus glycoprotein of apparent mol. wt. 60000. Preliminary studies showed that the antigen was expressed on all thymocytes and on peripheral B lymphocytes in smaller amounts. It was also present in brain, but not liver or kidney homogenate.     

Flow microfluorometric analysis of alloantigen expression during T cell development

The fluorescence-activated cell sorter (FACS) was used to examine the expression of several alloantigens on T cells: Thy-1, Lyt-1.1, Lyt-2.1, Ly-5, Ly-6 and Ly-7. Antibodies secreted by monoclonal cell lines were used to characterize the Thy-1.2, Lyt-1.1 and Lyt-2.1 antigenic determinants, whereas Ly-5.1, Ly-6.2 and Ly-7.2 determinants were identified with alloantisera raised by conventional hyperimmunization. Using indirect immunofluorescence with fluorescein isothiocyanate-conjugated reagents, the profiles obtained with the FACS demonstrated that: (a) the expression of Thy-1 is greater on normal thymocytes than on cortisone-resistant thymocytes (CRT), lymph node and spleen cells; (b) in contrast to Thy-1, the expression of Ly-1 on normal thymocytes is less than on CRT, lymph node and spleen cells; (c) about 90% of normal thymocytes but < 50% of CRT, spleen and lymph node T cells are Lyt-2⁺ and (d) although weakly expressed on normal thymocytes, the amount of Ly-6 and Ly-7 present on peripheral T cells was considerably increased. The significance of these findings and the potential for further analysis of T cell developmental pathways is discussed.     

The expression of IgE Fc receptors on lymphocytes of allergic patients

Peripheral blood lymphocytes from nonatopic subjects and atopic patients were analyzed for cells expressing Fc receptors for IgE (Fc epsilon R). Nonatopic humans and atopic patients in remission had approximately 1 percent of Fc epsilon R+ peripheral blood lymphocytes. Usually greater than 99 percent of these cells were mIgM+/mIgD+ B cells. However, in approximately 10 percent of nonatopic and atopic subjects a transient increase of Fc epsilon R+ lymphocytes to 3-6 percent was observed in the absence of any disease manifestations and measurable changes in the serum IgE level. At times of increased numbers of peripheral blood Fc epsilon R+ lymphocytes, up to 1 percent Fc epsilon R+ positive cells were detected in isolated T cell preparations. The Fc epsilon R+ T cells reacted with the monoclonal antibody Lyt 3 to the sheep erythrocyte receptor of human T cells but not the anti-T cell antibody OKT3, and fractions also with the monoclonal antibodies OKT8 (cytotoxic and suppressor T cells) and OKM1, which binds to an antigen present on monocytes and a subpopulation of T cells and large granular lymphocytes. No OKT4+ (helper T cells) Fc epsilon R+ cells were detected. The reactivity with monoclonal antibodies to T cell subsets of the Fc epsilon R+ T cells paralleled the reactivity of the IgG Fc receptor positive T cells. In contrast to patients with allergic rhinitis and asthma, patients with severe atopic dermatitis or the Hyper IgE Syndrome always had significantly elevated percentage of Fc epsilon R+ lymphocytes (4-10 percent), which were almost entirely B cells since less than 0.1 percent Fc epsilon R+ T cells were detected in these patients. Atopic dermatitis patients receiving systemic corticosteroid treatment had only 0.2 percent Fc epsilon R+ lymphocytes which was significantly less than the 1 percent of the nonatopic control donors. Attempts to define the function of Fc epsilon R on human B and T lymphocytes have been unsuccessful thus far; however, the increase of Fc epsilon R+ cells associated with atopic disease in man and parasitic infections in rats and mice suggest that Fc epsilon R+ lymphocyte may be involved in the IgE isotype regulation.     

T细胞亚群比例失调在血栓闭塞性脉管炎发病机制中的作用

关于血栓闭塞性脉管炎(TAO)的发病机制目前尚无定论。一些学者认为TAO的发生与机体免疫功能紊乱关系密切,主要表现在体液免疫功能亢进和细胞免疫功能低下,而T细胞在机体免疫反应的调控中可能起着主导作用。目前,有关TAO病人外周血T细胞亚群的研究,及其与抗动脉抗体(AAA)产生的关系,以及在疾病发生和发展中所起的作用,国内外报告甚少。本研究对     

Human T cells expressing V beta 8 do not predominantly recognize DR2 alloantigen.

A panel of seven monoclonal antibodies recognizing human T-cell antigen receptor (TcR) V alpha or V beta subsets has been used to measure TcR gene expression in peripheral blood lymphocytes and mixed lymphocyte culture responses (MLR) between DR2- and DR2+ (DRw15+) donors. There were no significant differences between DR2- and DR2+ donors in per cent T cells in fresh peripheral blood labelled with any of these antibodies, which included an antibody recognizing V beta 8. This indicates strongly that increased negative selection of V beta 8+ T cells does not occur in DR2+ compared with DR2- individuals. In MLR between DR2- and DR2+ donors the only significant change compared with fresh peripheral lymphocytes was that T cells expressing V beta 5.1 were decreased in DR2- lymphocyte populations responding to DR2 alloantigen. No changes in levels of V beta 8+ T cells were detected in MLR between DR2- and DR2+ donors. This suggests that V beta 8+ T cells are not predominantly reactive against DR2 (DRw15). The data support the concept that alloreactivity against a single class II major histocompatibility complex (MHC) mismatch is mediated by T cells expressing a range of different TcR V beta molecules.     

Mouse alloantibodies capable of blocking cytotoxic T cell function. II. Further study on the relationship between the blocking antibodies and the products of the Lyt-2 locus

The specificity of mouse alloantibodies which blocked allogeneic killer cells has been confirmed as Ly-2.2 using B 6 × C 3 H recombinant inbred (RI) strains. All of the RI strains tested were sensitive to the cell-mediated lymphocytotoxicity (CML)-blocking effect of our C3 H anti-B 10.BR antisera and also carried the Lyt-2^b allele. This result and studies using Lyt-2-congenic strains firmly localized the genes encoding the target antigen of CML-blocking antibodies in or near the Lyt-2 locus. In addition, a monoclonal anti-Lyt-2.2 antibody was found to block the CML reaction of B 6 killer cells, but not that of B 6 Ly-2.1 killers. Blocking antibodies to one allele blocked killing by Lyt-2-heterozygous F₁ killer cells, but the inhibition curve reached a plateau at a significantly lower level than that seen on the killer cells of the sensitive parent. Our results thus suggest that Lyt-2 antigens are of functional significance on killer T cells, but do not reveal whether or not that significance involves the T cell receptor.     

Selective up-regulation by interferon-gamma of surface molecules of the Ly-6 complex in resting T cells: the Ly-6A/E and TAP antigens are preferentially enhanced

Surface molecules encoded by the murine Ly-6 locus can transduce triggering signals in T cells and thus may play important roles in T cell function. Previously, we found that Ly-6 molecules are up-regulated by interferon (IFN)-alpha/beta in resting T cells. Here, we examined the possible influence of IFN-gamma on these molecules. Purified T cells from C57BL/6 (Ly-6.2) and BALB/c (Ly-6.1) mice were incubated in vitro with recombinant murine IFN-gamma and the expression of Ly-6 antigens was measured by flow cytofluorometry. It was found that both Ly-6A/E and T cell-activating protein (TAP) molecules are markedly enhanced while Ly-6C is less affected. Under the same conditions, other T cell surface molecules showed no or marginal changes. The effect of IFN-gamma on Ly-6A/E and TAP expression reached a maximum with as little as 10 U/ml and required only 18-24 h of incubation. Moreover, the enhancement of Ly-6A expression induced by IFN-gamma was stable for at least 5 days. Analysis of T cell subsets further revealed that IFN-gamma-induced augmentation of Ly-6A (C57BL/6 mice) involves both Lyt-2+ and L3T4+ cells while the increase of Ly-6E (BALB/c mice) is more pronounced in Lyt-2+ cells. The functional consequence of these phenotypic alterations was evaluated by studying the mitogenic responses of T cells to antibody-mediated Ly-6 cross-linking in the presence of phorbol myristate acetate. Pretreatment of resting T cells with IFN-gamma dramatically increased the responses to anti-Ly-6A and anti-Ly-6E monoclonal antibodies. IFN-gamma treatment also boosted the stimulation induced by anti-TAP monoclonal antibody when this stimulation was performed under suboptimal conditions. Therefore, IFN-gamma selectively up-regulates the Ly-6A/E and TAP activation pathways in resting T cells. We speculate that this effect may contribute to the immunoregulatory activities of IFN-gamma.     

Generation of a monoclonal antibody that recognizes a polymorphic epitope of C57BL/6 Ly-49G2

To facilitate the study of natural killer (NK) cell functions, we have generated a panel of hybridomas that secrete antibodies recognizing B6 NK cell surface markers. One of these hybridomas, Cwy-3, secretes a mouse IgG1 specific for the B6 allele of Ly-49G2 (Ly-49G2B6). In addition, this monoclonal antibody (MAb) fails to stain A-LAKs derived from all other mouse strains tested. This suggests that Cwy-3 recognizes a polymorphic epitope of Ly-49G2B6. More importantly, this MAb exhibits no cross-reactivity to other known B6 Ly-49 family members. Therefore, Cwy-3 is in sharp contrast to the two known anti-Ly-49G2 MAbs, which are reported to recognize a nonpolymorphic epitope of Ly-49G2, and react with other Ly-49 members. In this regard, Cwy-3 will offer significant advantages over the cross-reactive anti-Ly-49G2 MAbs in fully defining the specificity and function of the Ly-49G2B6 NK cell receptor. With this novel Ly-49G2-specific MAb, we have isolated an EL-4 subline expressing a significant density of Ly-49G2 receptors.     

The mitogenic lectin from Phaseolus vulgaris does not recognize the T3 antigen of human T lymphocytes

Human peripheral blood T lymphocytes are stimulated to grow and divide by lectins such as concanavalin A (Con A) and Phaseolus vulgaris phytohemagglutinin (PHA), as well as a few anti-T cell monoclonal antibodies. The latter antibodies recognize the T3 antigen. It has been suggested previously that PHA and Con A mediate T cell growth by interacting with T3. However, as reported in this study, affinity chromatography on immobilized lectins, and immunoprecipitation by lectin plus anti-lectin antibodies showed that T3 binds Con A but not PHA. Fab fragments of a monoclonal antibody against T3 (namely Leu-4) inhibited T lymphocyte proliferation induced by T3 antibodies and Con A, but not by PHA. Nevertheless, co-capping experiments performed with fluorescein-labeled lectins and rhodamine-labeled T3 antibodies showed that T3 co-caps with Con A and PHA receptors, although the co-capping with PHA was incomplete. Since the T cell receptor for antigen (Ti) has been shown to co-cap with T3 on the cell surface, we reasoned that PHA induced capping of the T3 antigen by interacting with Ti. A disulfide-linked heterodimer comprising subunits of about 49 000 and 41 000 mol. wt. that resembled the Ti molecule was detected in PHA-anti-PHA immunoprecipitates of various surface- and biosynthetically-labeled T cells, by two-dimensional (nonreduced vs. reduced) sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. The results suggest that PHA triggers T lymphocytes by interacting with the carbohydrate moieties of Ti and imply that T lymphocytes can be stimulated by mitogens via at least two different cell surface molecules (Ti and T3).     

Major histocompatibility complex class I-dependent cell binding to isolated Ly-49A: evidence for high-avidity interaction

Ly-49A molecules negatively regulate a subset of mouse natural killer (NK) cells, preventing lysis of H-2D^d-expressing target cells. In the present report, we immunoaffinity-purified Ly-49A from the EL4 lymphoma using the A1 monoclonal antibody (mAb) and examined cell adhesion to immobilized Ly-49A. Adhesion was observed by cells expressing relatively high levels of H-2D^d, but not cells expressing very low or no cell surface D^d, while antibodies specific for D^d or Ly-49A inhibited the cell binding, indicating that D^d and Ly-49A mediate the observed adhesion. The density of immobilized Ly-49A was varied and confirmed by ELISA. Cell binding exhibited a threshold Ly-49A density requirement, and above this threshold, increases in Ly-49A density resulted in substantial increases in cell adhesion to a high maximum cell binding. The density of Ly-49A homodimers required to mediate cell adhesion was found to be quite low: 140–250 molecules/μm². These results suggest that the avidity of Ly-49A for D^d is relatively high and indicate that small changes in Ly-49A density near the threshold result in large changes in stable Ly-49A receptor engagement. The relatively sharp threshold and marked density dependence presented here for Ly-49A receptor engagement may explain the observation that relatively small differences in Ly-49A expression level on NK cells result in significant differences in functional outcome, i.e. whether a target cell expressing a low level of D^d is spared from lysis or not.