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The aim was to establish an in vitro model for studies of innate defence mechanisms of human intestinal epithelium. Ultrastructural characterization and determination of mRNA expression levels for apical glycocalyx and mucous components showed that polarized, tight monolayers of the colon carcinoma cell lines T84 and Caco2 acquire the features of mature- and immature columnar epithelial cells, respectively. Polarized monolayers were challenged with non-pathogenic Gram+ and Gram- bacteria from the apical side and the proinflammatory cytokines interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) from the basolateral side. Immune responses were estimated as changes in mRNA expression levels for the mucous component mucin-2 (MUC2), the glycocalyx components carcinoembryonic antigen (CEA), CEA-related cell adhesion molecule-1 (CEACAM1), CEACAM6, CEACAM7 and MUC3, the antimicrobial factors human beta-defensin-1 (hBD1), hBD2, hBD3 and lysozyme, the chemokine IL-8 and the cytokines IL-6 and TNF-alpha. Tight monolayer cells were generally unresponsive to bacterial challenge, but increased their hBD2 levels when challenged with Bacillus megaterium. T84 cells also increased their TNF-alpha levels upon bacterial challenge. Tight monolayer cells responded to cytokine challenge suggesting awareness of basolateral attack. TNF-alpha induced significantly increased levels of IL-8 and TNF-alpha itself in both cell lines suggesting recruitment and activation of immune cells in the underlying mucosa in vivo. Cytokine challenge also increased levels of CEACAM1, which includes two functionally different forms, CEACAM1-L and CEACAM1-S. In T84 cells, IFN-gamma was selective for CEACAM1-L while TNF-alpha upregulated both forms. Increased CEACAM1 expression may influence epithelial function and communication between epithelial cells and intraepithelial lymphocytes.

Cell-mediated lymphocyte cytotoxicity in ileum and colon of patients with ulcerative colitis (UC), Crohn's disease (CD) and controls was investigated. Frequencies of cells expressing perforin and Fas-ligand (FasL) were determined by immunomorphometry. mRNA expression of perforin, granzyme B and FasL in T cells and subsets was assayed by reverse transcriptase-polymerase chain reaction. Cytotoxicity of intraepithelial and lamina propria lymphocytes was analysed without ex vivo activation in three functional assays: (1) anti-CD3-dependent T-cell receptor (TCR)-/CD3-mediated redirected cytotoxicity, (2) Fas-/FasL-mediated TCR-/CD3-independent cytotoxicity and (3) natural killer (NK) cell cytotoxicity. Inflammation in ileum of CD patients caused increased frequency of perforin-expressing cells and enhanced perforin-dependent TCR-/CD3-mediated cytotoxicity. In contrast, lymphocytes in the inflamed colon of UC or Crohn's colitis patients did not display this cytotoxicity nor did lymphocytes of normal colon. Normal colon lymphocytes showed spontaneous Fas-/FasL-mediated cytotoxicity. This activity was retained but not enhanced in inflamed UC colon. In contrast, a significant increase of FasL-expressing cells was seen in situ. Inflammation did not induce NK cell activity in colonic lymphocytes. Intestinal lymphocytes comprise effectors active in two different cytolytic processes. 'Classical' cytotoxic T lymphocytes in small intestine and lymphocytes executing TCR-/CD3-independent FasL-/Fas-mediated killing of unknown biological role present throughout the intestinal mucosa. Ongoing normal cytolytic processes seem to be enhanced by chronic inflammation.

The accuracy of 18S rRNA, beta-actin mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as indicators of cell number when used for normalization in gene expression analysis of T lymphocytes at different activation stages was investigated. Quantitative real-time reverse transcriptase-polymerase chain reaction was used to determine the expression level of 18S rRNA, beta-actin mRNA, GAPDH mRNA and mRNA for six cytokines in carefully counted samples of resting human peripheral blood mononuclear cells (PBMCs), intestinal lymphocytes and PBMCs subjected to polyclonal T-cell activation. The 18S rRNA level in activated and resting PBMCs and intestinal lymphocytes was essentially the same, while the levels of beta-actin and GAPDH mRNAs fluctuated markedly upon activation. When isolated gammadeltaTCR(+), CD4(+) and CD8(+) subpopulations were studied, 18S rRNA levels remained unchanged after 21 h of activation but increased slightly after 96 h. In contrast, there was a 30-70-fold increase of GAPDH mRNA/cell in these cell populations upon activation. Cytokine analysis revealed that only normalization to 18S rRNA gave a result that satisfactorily reflected their mRNA expression levels per cell. In conclusion, 18S rRNA was the most stable housekeeping gene and hence superior for normalization in comparative analyses of mRNA expression levels in human T lymphocytes.

Four carcinoembryonic antigen-related cell adhesion molecule (CEACAM)s, i.e. CEA, CEACAM1, CEACAM6 and CEACAM7, are localized to the apical glycocalyx of normal colonic epithelium and have been suggested to play a role in innate immunity. The expression of these molecules in colon carcinoma cells was studied at the mRNA and protein levels after treatment with interferon-gamma (IFN-gamma), interleukin-1beta, live bacteria or lipopolysaccharide. The colon carcinoma cell lines LS174T and HT-29 were studied in detail using real-time quantitative reverse transcriptase-polymerase chain reaction, immunoflow cytometry and immunoelectron microscopy. IFN-gamma, but not the other agents, modified expression of CEA, CEACAM1 and CEACAM6. None of the agents upregulated CEACAM7 expression. Two expression patterns were seen. HT-29 cells, which initially showed low quantities of mRNAs and proteins, displayed marked upregulation of both mRNAs and proteins. LS174T cells transcribed stable high levels of mRNA before and after treatment. Additionally, IFN-gamma induced increased cell surface expression of CEA, CEACAM1 and CECAM6. IFN-gamma has two important effects on the expression levels of the CEA family molecules in colon epithelial cells: direct upregulation of CEACAM1 and promotion of cell differentiation resulting in increased expression of CEA and CEACAM6 and decreased expression of CEACAM7.

Intraepithelial lymphocytes (IEL) in normal human small intestine exhibit cytotoxicity. This study was undertaken to characterize the effector cells and their mode of action. Freshly isolated jejunal IEL and lamina propria lymphocytes (LPL), as well as IEL and LPL depleted of CD4+, CD8+ and T-cell receptor (TCR)-gammadelta+ cells were used as effector cells in anti-CD3-mediated redirected cytotoxicity against a murine FcgammaR-expressing cell line. Effector cell frequencies were estimated by effector to target cell titration and limiting dilution. The capacity of IEL and LPL to kill a Fas-expressing human T-cell line was also analysed. T-cell subsets were analysed for perforin, granzyme B, Fas-ligand (FasL), tumour necrosis factor-alpha (TNF-alpha) and TNF-related apoptosis inducing ligand (TRAIL) mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR). Frequencies of IEL expressing the perforin and FasL proteins were determined by immunomorphometry. Both IEL and LPL exhibited significant Ca2+-dependent, anti-CD3-mediated cytotoxicity, approximately 30% specific lysis at the effector to target cell ratio 100. The cytotoxic cells constituted, however, only a small fraction of IEL and LPL ( approximately 0.01%). CD8+ TCR-alphabeta+ cells accounted for virtually all the cytotoxicity and expressed mRNA for all five cytotoxic proteins. The frequency of granzyme B-expressing samples was higher in CD8+ cells than in CD4+ cells (P<0.05 and <0.01 for IEL and LPL, respectively). In addition, both IEL and LPL exhibited significant spontaneous anti-CD3-independent cytotoxicity against Fas-expressing human T cells. This killing was mediated by Fas-FasL interaction. On average, 2-3% of the IEL expressed perforin and FasL. We speculate that CD8+ memory cells accumulate in the jejunal mucosa and that the CD8+ TCR-alphabeta+ lymphocytes executing TCR/CD3-mediated, Ca2+-dependent cytotoxicity are classical cytotoxic T lymphocytes 'caught in the act' of eliminating infected epithelial cells through perforin/granzyme exocytosis. The observed Fas/FasL-mediated cytotoxicity may be a reflection of ongoing down-regulation of local immune responses by 'activation-induced cell death'.

Lactoferrin (Lf), an iron-binding protein in milk, mucosal secretions and neutrophil granules has bactericidal properties and is a source of iron for breast-fed infants. In this paper the authors show that most in vivo activated lymphocytes, i.e. freshly isolated lymphocytes from first trimester human decidua, and most in vitro activated human blood lymphocytes, express lactoferrin receptors (Lf-R), while unstimulated blood lymphocytes do not. All major lymphocyte subsets, i.e. alpha beta T cells, gamma delta T cells, CD8+ T cells, CD4+ T cells, B cells and NK cells, express Lf-R after activation. The proportion of Lf-R expressing activated gamma delta T cells is significantly larger than that of activated alpha beta T cells. Lf-R and transferrin receptors (Tr-R/CD71) show the same kinetics of appearance on activated blood lymphocytes and are, to a large extent, expressed on the same cells. However, 35% of decidual lymphocytes and 15% of activated blood lymphocytes express Lf-R only. Addition of Lf to cultures containing an optimal concentration of Tr augments the proliferative response to polyclonal T cell activators and alloantigens, suggesting that presently used standard culture conditions for in vitro activation are suboptimal in particular for gamma delta T cells. Lf-R on decidual lymphocytes contain bound Lf, which probably is produced locally. The results suggest that Lf is a growth-supporting factor, especially important in local immune responses in the mucosa.

The proliferative response induced by leucoagglutinin (La) in different subpopulations of human T lymphocytes was studied. Subpopulations enriched in cells with either high- or low-avidity receptors for sheep erythrocytes (SRBC) (EH +, EL +) were prepared by sequential E-rosetting. In addition, T lymphocytes prepared by E-rosetting under optimal conditions (E + TOT) were fractionated on wheat germ agglutin (WGA)-Sepharose columns, rendering fractions enriched in lymphocytes with either low- or high-avidity receptors for WGA. The T lymphocytes were found to comprise at least three functionally distinct subpopulations, differing with respect to mitogen responsiveness. Cells characterized by high-avidity receptors for WGA and SRBC were highly responsive to La stimulation, regardless of the method used for purification. In contrast, cells with low-avidity receptors for WGA and probably also for SRBC but lacking Fc receptors for IgG responded only marginally but were conditioned to respond when subjected to E-rosetting under optimal conditions. This response was suppressed by lymphocytes with Fc receptors for IgG, which probably also had low-avidity receptors for WGA and SRBC. The lymphocytes with high-avidity receptors for WGA ans SRBC did not appear to be susceptible to suppression by Fc gamma + cells.

The relationship between the surface receptors on neuraminidase-treated human blood lymphocytes for the mitogenic lectins Phaseolus vulgaris leukoagglutinin (La), concanavalin A (Con A) and soy bean agglutinin (SBA) and the non-mitogenic lectin Helix pomatia A hemagglutinin (HP) was investigated. Two different techniques, co-capping with different fluorochrome-labeled lectins and cell binding-inhibition experiments with 125I-labeled lectins, were used. The results demonstrated that the nonmitogenic lectin HP and the mitogenic lectins SBA, La and Con A bind either to the same macromolecule (s) or to different but physically linked macromolecules on the surface of human T lymphocytes. In contrast, only part of beta2-microglobulin (beta2-m) or beta2-m-bearing complexes, appear to be physically linked to the lectin receptor complex(es). On the lectin-binding substance(s) at least two saccharide structures were recognized, one of which binds both HP and SBA and another which binds SBA and La (and probably also Con A) but not HP.

Activation of human peripheral blood lymphocytes (PBL) with the mitogenic monoclonal antibody (MoAb) K46M, which recognizes 1-5% of PBL, resulted in the expansion of cells with cytolytic activity. Thus, after culture of the activated lymphocytes in medium containing interleukin 2 (IL-2), they lysed a variety of cultured cell lines. The majority of the activated lymphocytes reacted with MoAb to CD8, CD3, and to the T cell antigen receptor heterodimer (Ti) but not with antibodies to antigens expressed on natural killer (NK) cells. The cytotoxicity was not inhibited by MoAb to CD3 or Ti. However, the killing of K562, but not of other cell lines, was enhanced by three to four times in the presence of anti-Ti antibodies. Anti-CD3 or other control antibodies had no effect. Cold target inhibition experiments indicated that the cytolytic lymphocytes recognized closely related structures on the target cells. Phenotypically and functionally similar effector cells emerged after activation of PBL with the anti-CD3 MoAb OKT3. Taken together, the results indicate that activation of PBL with MoAb K46M induces cytotoxic cells that differ from classical NK cells but that resemble mature cytotoxic T lymphocytes (CTL). However, unlike CTL, cytotoxicity was not MHC-restricted and the conventional T-cell receptor complex (CD3/Ti) appeared not to be involved in target cell recognition and cytolysis.

Fluorochrome-labelled Helix pomatia A haemagglutinin (HP) stained T-dependent areas in cryostat tissue sections of human lymph node, tonsil, and spleen and thymocytes within thymic medulla but not within thymic cortex. Neuraminidase treatment of the tissue section was a prerequisite for the positive cell staining. The staining was cell-membrane-associated, and the histological pattern was essentially the same as that observed in parallel sections treated with monoclonal antibodies to T cells (OKT3, Leu-1). In addition, blood vessel walls and connective tissue were also stained by HP conjugates. The latter staining was independent of neuraminidase treatment, resistant to alpha-galactosidase treatment and was not blocked by pretreatment with anti-fibronectin antibody. Body types of staining reaction were specific inasmuch as unlabelled HP and the competitive sugar hapten N-acetyl-D-galactosamine blocked the reaction, Frozen, formalin-fixed tissue sections showed the same stainability and reaction pattern as unfixed sections.

A monoclonal antibody, K46M (IgM kappa), obtained after immunization with leucoagglutinin (La)-reactive T-cell surface components, stimulated human lymphocytes to proliferate. It induced maximal proliferation at greater than 20 micrograms IgM/ml after 3-4 days of culture. Cells stimulated by K46M produced interleukin 2 (IL-2) and gamma interferon (IFN-gamma) and expressed receptors for IL-2 and transferrin. The majority of the activated cells were phenotypically T cells as defined by monoclonal antibodies against CD3 and CD2, and an increase in the K46M-positive cells was also observed during the activation period. K46M-activated cells display major histocompatibility complex (MHC)-unrestricted cytotoxicity against several cultured target cells. The frequencies of the cytotoxic and of the proliferative precursor cells were determined using a limiting dilution assay. K46M seems to activate a larger fraction of cytotoxic precursor cells against Molt 4 than against K562, but the statistical significance of these observations requires further exploration. Both K46M or La activated 40% of PBL to proliferate, whereas 70% of PBL were induced by OKT3. However, the frequency of K46M-activated cells was 40% only when the lymphocytes were plated at low cell densities, i.e. less than 0.5 cells per well. At higher densities an inhibition of proliferation was seen that resulted in a biphasic response curve, indicating that the activation of PBL by K46M was not a single hit event. This was not found with either La or OKT3. Whether K46M, in contrast to OKT3 and La, activates a subpopulation with suppressor activity remains to be established.

The leukotrienes (LT's) are a group of arachidonic acid derivatives implicated as mediators of allergic bronchoconstriction and acute inflammation. Tracheal spirals and strips of lung parenchyma from guinea pigs were used under non-flow conditions to characterize the contractions caused by LTA4, LTB4 and LTC4. Cumulative administrations of leukotrienes desensitized the lung strip, whereas non-cumulative dose-response relationships for the leukotrienes and histamine were reasonably parallel. Half maximal contractions of the lung strips were obtained at a final bath concentration of 1 nM for LTC4 and 300 nM for LTA4 or LTB4, as compared with 6 000 nM for histamine. In the trachea, LTC4 was approximately 100 times more potent than LTA4 and histamine. Leukotrienes B4 and C4, but not acetylcholine or histamine, elicited release of the bronchoconstrictive thromboxane A2 from the lung under non-flow conditions. Indomethacin blocked the contractile response to LTB4, whereas the contractile effect of LTC4 remained unaltered. The beta-adrenoceptor agonist isoproterenol and the LTC4 antagonist FPL 55712 attenuated the contraction, but not the release of thromboxane A2, induced by LTC4. Changing to a perifusion technique rendered the lung strips less sensitive to the direct action of LTC4, and released thromboxane A2 now contributed significantly to the contractile response. In addition, the perifusion experiments indicated that LTB4 released histamine as well. We conclude that the chemoattractant LTB4 is an indirectly acting bronchoconstrictor, whereas the slow reacting substance LTC4 contracts the airway muscle by a predominantly direct mechanism. The exquisite bronchoconstrictive activity of LTC4 may be unrelated to its ability to induce formation of thromboxane A2.

The appearance of transferrin receptors on mitogen-stimulated human thymus-derived (T) lymphocytes was studied. When indirect immunofluorescence with immunoadsorbent-purified antitransferrin antibodies was used, approximately 10% of resting T cells were stained. This proportion increased to 50-80% of the cells 3-4 days after stimulation with the mitogenic lectins concanavalin A (Con A) and leucoagglutinin (La) from Phaseolus vulgaris. Almost all blast cells (greater than or equal to 90%) were positive. Cell binding experiments with 125I-labelled transferrin indicated the presence of 1-5 x 10(5) transferrin receptor molecules/cell with high avidity for transferrin (K = 2 - 12 x 10(8) l/mol). Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography of cell lysates containing 125I-labelled T-cell surface components revealed two surface peptides (90 kdaltons and 80 kdaltons, reducing conditions), which selectively bound to insolubilized antitransferrin antibodies. The 90-kdalton peptide also bound to insolubilized transferrin. The 80-kdalton peptide is most probably transferrin and the 90-kdalton peptide the transferrin receptor. Unreduced transferrin receptor had a molecular weight of 180 kdalton. It is probably a glycoprotein, since it reacted with wheat germ agglutinin, La, and probably also Con A. The properties of the lymphocyte transferrin receptor are similar to those described for transferrin receptors on various in-vitro-grown transformed cells. This speaks in favour of a common receptor present on all proliferating human cells.

Human T-lymphocyte preparations from peripheral blood with either high- or low-avidity receptors for wheat germ agglutinin (WGA) were obtained by fractioned on WGA--Sepharose columns. Both fractions contained progenitors of alloreactive T cells, proliferating in mixed lymphocyte culture (MLC) and acting as effector cells in cell-mediated lympholysis (CML). Proliferation and CML activity of the two fractions were equal and similar to those of the unfractionated cells. However, when the lymphocytes were fractionated after 5 days' MLC, most of the proliferating and cytolytic cells were found in the lymphocyte fraction enriched in cells with high-avidity receptors for WGA. The reactivity of the fractions was correlated to their content of blast-transformed cells. The binding of MLC-activated lymphocytes to WGA was specific, since it was inhibited by the competitive hapten D-GlcNAc and no cells were retained on control columns charged with human serum albumin-Sepharose. B cells and monocytic cells were enriched in the fraction with low-avidity receptors for WGA. As indicated by experiments in which cells were mixed in different proportions, the low MLC-CML activity of the lymphocytes in the fraction with low-avidity WGA receptors was not caused by suppressor cells present in that fraction.

Thirty-two percent of neuraminidase-treated DA rat spleen lymphocytes and 48% of lymph node lymphocytes possess receptors for Helix pomatia hemagglutinin (HP). Moreover, these HP-receptor-bearing cells can be separated from B cells by affinity chromatography on HP-Sepharose columns. The virtual absence of immunoglobulin (Ig) receptors and the close correlation with reported T-cell content of these lymphoid tissues suggest that HP-receptor lymphocytes are probably T cells and that HP may provide a convenient marker, for both the identification and the purification of rat T lymphocytes.

The effect of polyclonal (anti alpha and beta chain) and monoclonal (anti alpha-chain) antibodies against lymphocyte function-associated antigen 1 (LFA-1) on T cell activation was studied. When added at the beginning of activation but not after 24 h or later the antibodies as well as the F(ab')2 or Fab fragments of polyclonal antibodies inhibited concanavalin A (Con A)-induced proliferation, interleukin 2 (IL-2) production, and the expression of receptors for IL-2 and transferrin. The inhibitory effect reached a maximum at the same time as optimal proliferation (72 h). Inhibition of proliferation lasted for 5 days or longer, although IL-2 production was only inhibited during the first 48 h of culture. Receptors for IL-2 and transferrin were re-expressed to the original level after 3 days of activation. Addition of external IL-2 at the beginning of the anti-LFA-1 containing culture prevented the inhibition of IL-2 receptor expression, while inhibition of transferrin receptor expression was unaffected, supporting the conclusion that the expression of these two receptors is regulated by partially independent signals. The polyclonal and monoclonal anti-LFA-1 antibodies also inhibited phorbol ester (PMA)-dependent OKT3 activation of highly purified T cells. The results suggest that the LFA-1 antibodies block an early step in the reactions necessary for IL-2 production, and that the LFA-1 molecule participates not only in T cell-accessory cell interaction but also in T-T interaction during the early phases of the activation process.

Human peripheral blood lymphocytes (PBL) were activated with K46M, a m mitogenic monoclonal antibody against La-reactive T lymphocyte surface structures. The cultures were expanded in the presence of interleukin 2 (IL-2). After 1 month of culture, the activated T cells were cloned by limiting dilution at 0.5 cells/well. Five clones with the CD3+CD4+ phenotype and one clone with the CD3+CD8+ phenotype were obtained. The CD3+CD8+ clone (K99) displayed a strong major histocompatibility complex (MHC)-unrestricted cytolytic activity against MOLT-4 and a weaker reactivity against the bladder tumour cell lines T24 and RT4. The natural killer (NK)-susceptible K562 cells were not lysed. Two of the CD3+CD4+ clones (K91 and K914) showed a helper activity in pokeweed mitogen (PWM)-induced IgG production by B cells. These cells differed in the expression of CD45R and CDw29 antigens, as defined by the monoclonal antibodies 2H4 or D10D11 and 4B4. When stimulated with PWM for 48 or 72 h, clone K91 and an additional CD4-positive clone (K913) secreted a factor into the supernatants which helped B cells to produce IgG. The K913 supernatant also induced some IgM production. The supernatant obtained after similar stimulation of K914 cells was inactive. None of these supernatants induced B cells to proliferate when tested together with phorbol myristate acetate (PMA). However, when K91 and K914 cells were activated with phytohaemagglutinin (PHA) for 48 or 72 h, the supernatant from K91 was strongly helpful in B-cell proliferation, whereas the supernatant from K914 cultures was only moderately active. In conclusion, we have established human T helper clones that release different factors supporting either B-cell proliferation or maturation when stimulated with PWM or PHA.

Treatment of human blood lymphocytes with neuraminidase has previously been shown to uncover receptors for the A haemagglutinin of the snail Helix pomatia (HP). Neuraminidase-treated lymphocytes were now fractionated on columns charged with large Sepharose particles to which HP had been coupled covalently. HP-receptor negative (HP-) lymphocytes passed the columns while HP-receptor positive (HP+) lymphocytes were retained. The latter cells were eluted by addition of the competitive hapten N-acetyl-D-galactosamine (D-GalNAc). The total yield of cells recovered after fractional was 60-80% Surface marker studies indicated that there was no selective loss of any of the major lymphocyte subpopulations. The fraction that passed the columns (fraction I) consisted of approximately 10% of all lymphocytes. It contained approximately 1% HP+cells and approximately 3% of all lymphocytes forming rosettes which sheep erythrocytes (E+ cells) present before fractionation. 50-55% of the lymphocytes in this fraction had surface-bound immunoglobulin (SIg+ cells) and complement receptors (EAC+ cells). Of the SIg+ cells, approximately 60% were true B cells while the remaining 40% had IgG adsorbed to their surface. The majority of the B cells were recovered in this fraction. The lymphocytes of this fraction responded poorly to T-cell mitogen but had an enhanced K-cell activity to chicken erythrocytes. Elution of the cells retained on the column with 0.1 mg/ml D-GalNAc gave a fraction II, consisting of approximately 15% of all lymphocytes. This fraction had a mixed composition. The majority of the cells (approximately 45%) were recovered by subsequent elution with 1.0 mg/ml D-GalNAc. This fraction III was strongly enriched with HP+ and E+ cells (T cells). About 10% of the HP+ cells in this fraction were SIg+. However, on the majority of these cells this surface-bound immunoglobulin was probably externally absorbed IgG. These HP+-SIg" cells were also EAC+ and had Fc receptors, as shown by rosette formation with IgG-coated bovine erythrocytes. The lymphocytes of fraction III responded most strongly to T-cell mitogen while their K-cell activity was weak.

Receptors for Helix pomatia A haemagglutinin (HP) have earlier been found on neuramidinase treated T-lymphocytes in human peripheral blood. In contrast, the majority of the B-lymphocytes, characterized by surface bound IgM and/or IgD(SIg)lack these receptors. Double marker experiments with fluorochrome labelled reagents have now shown that a minor fraction (3--24%) of the IgM/d bearing lymphocytes in normal human blood also have HP-receptors. These HP+ B-cells constitute approximately 1% of the HP+ lymphocytes in adult blood. Fractionation on HP-Sepharose columns showed that the HP+ B-cells are readily eluted with buffer containing 0.1 mg N-acetyl-D-galactosamine. In contrast, the majority of the HP+-SIg--cells require higher concentrations of the competing hapten for elution (1 mg D-Ga1NAc/ml buffer). This indicates that the HP-receptors on these B-cells differ qualitatively or quantitatively from those on the majority of the T-cells. Previous findings of HP-receptors on the SIg+ leukaemic cells in the blood of patients with chronic lymphocytic leukemia suggested that these structures are expressed on an immature variety of B-cells. This assumption is favoured by the present finding that approximately 80% of the lymphocytes with surface bound IgM/D in cord blood also have HP-receptors. Therefore, the HP-receptors seems to fall in the category of differentiation markers and constitutes a useful tool for characterization and separation of human lymphocytes within both the T- and the B-compartments.

The binding specificities of three biologically active anti-lymphocyte monoclonal antibodies (MoAb) (K46M, K3G, and 3-19-2) produced against human T-cell surface components reactive with the mitogenic lectin leucoagglutinin from Phaseolus vulgaris (La) were analysed. K46M is a strong T-cell mitogen, while K3G and 3-19-2 inhibited cell-mediated cytotoxicity. Resting peripheral blood lymphocytes (PBL) contained 4-16% K46M+ cells, 8-35% K3G+ cells, and less than 0.3-4% 3-19-2+ cells. After stimulation with T-cell mitogens the proportion of K46M+ and 3-19-2+ cells increased markedly (mean 59 and 30% positive cells, respectively), while the increase in K3G+ cells was less prominent (38%). K46M-reactive structures were expressed on mature T cells and probably also on B cells. K3G reacted with B and T cells while 3-19-2 showed a broader specificity reacting also with erythrocytes. All three MoAb reacted with lipid extracts of resting and activated PBL as well as with purified neutral glycolipids of lymphoid origin. In addition 3-19-2 reacted with lipid extracts of erythrocytes. K46M immuno-precipitated four surface peptides from lectin-stimulated PBL. Their apparent molecular weights were 53,000, 42,000, and 16,000 (doublet). The 53,000 and 42,000 MW peptides were identified as the alpha and beta chains of the T-cell antigen receptor. The identity of the 16,000 MW peptides is presently unknown. K3G and 3-19-2 did not specifically precipitate any lymphocyte surface peptide.