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A hallmark of active celiac disease (CD), an inflammatory small-bowel enteropathy caused by permanent intolerance to gluten, is cytokine production by intestinal T lymphocytes. Prerequisites for contracting CD are that the individual carries the MHC class II alleles HLA-DQ2 and/or HLA-DQ8 and is exposed to gluten in the diet. Dysbiosis in the resident microbiota has been suggested to be another risk factor for CD. In fact, rod shaped bacteria adhering to the small intestinal mucosa were frequently seen in patients with CD during the "Swedish CD epidemic" and bacterial candidates could later be isolated from patients born during the epidemic suggesting long-lasting changes in the gut microbiota. Interleukin-17A (IL-17A) plays a role in both inflammation and anti-bacterial responses. In active CD IL-17A was produced by both CD8(+) T cells (Tc17) and CD4(+) T cells (Th17), with intraepithelial Tc17 cells being the dominant producers. Gluten peptides as well as CD associated bacteria induced IL-17A responses in ex vivo challenged biopsies from patients with inactive CD. The IL-17A response was suppressed in patients born during the epidemic when a mixture of CD associated bacteria was added to gluten, while the reverse was the case in patients born after the epidemic. Under these conditions Th17 cells were the dominant producers. Thus Tc17 and Th17 responses to gluten and bacteria seem to pave the way for the chronic disease with interferon-γ-production by intraepithelial Tc1 cells and lamina propria Th1 cells. The CD associated bacteria and the dysbiosis they might cause in the resident microbiota may be a risk factor for CD either by directly influencing the immune responses in the mucosa or by enhancing inflammatory responses to gluten.

Background: Coeliac disease is a small intestine enteropathy caused by permanent intolerance to wheat gluten. Gluten intake by patients with coeliac disease provokes a strong reaction by intestinal intraepithelial lymphocytes (IELs), which normalises on a gluten-free diet., Aim: To investigate whether impaired extrathymic T cell maturation and/or secondary T cell receptor (TCR) gene recombination in IELs are features of coeliac disease which could contribute to the failure of establishing tolerance to gluten., Methods: Expression levels of the four splice-forms of recombination activating gene-1 (RAG1) mRNA and preT alpha-chain (preTalpha) mRNA were determined in IEL-subsets of children with coeliac disease and controls. Frequencies of RAG1 expressing IELs were determined by immunomorphometry., Results: In controls, the RAG1-1A/2 splice-form selectively expressed outside the thymus, was dominant and expressed in both mature (TCR(+)) and immature (CD2(+)CD7(+)TCR(-)) IELs ( approximately 8 mRNA copies/18S rRNA U). PreTalpha was expressed almost exclusively in CD2(+)CD7(+)TCR(-) IELs ( approximately 40 mRNA copies/18S rRNA U). By contrast, RAG1 and preTalpha mRNA levels were low in patients with coeliac disease compared to controls, both with active disease and with inactive, symptom-free disease on a gluten-free diet (p values <0.01 for mature and <0.05 for immature IELs). Similarly, the frequencies of RAG1+ IELs were significantly lower in patients with coeliac disease compared to controls (p<0.001)., Conclusions: Patients with coeliac disease appear to have an impaired capacity for extrathymic TCR gene rearrangement. This is an inherent feature, which probably plays a pivotal role in the failure to efficiently downregulate the T cell response to gluten.

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.

Background & Aims: Specific T-lymphocyte reactions are central in the pathogenesis of celiac disease, an inflammatory small-bowel enteropathy caused by a permanent intolerance to gluten. To delineate local T-lymphocyte responses to gluten, the cytokine expression in jejunal T lymphocytes of pediatric celiac patients with active disease, i.e., untreated and gluten-challenged celiac patients, was determined and compared with that of treated, symptom-free celiac patients and controls., Methods: Biopsy samples were collected from celiac patients and controls. Intraepithelial and lamina propria T lymphocytes were isolated separately, and the cytokine messenger RNA levels were determined by using quantitative real-time reverse-transcription polymerase chain reaction. Interferon (IFN)-gamma and interleukin (IL)-10 were determined at the protein level by immunohistochemistry., Results: Active celiac disease was characterized by distortions in cytokine expression by T lymphocytes, with highly significant increases of IFN-gamma and IL-10 but no concomitant increases in tumor necrosis factor alpha, transforming growth factor beta1, or IL-2 and no induction of IL-4. A marked shift of IFN-gamma and IL-10 production from the lamina propria to the epithelium was characteristic of active celiac disease, and as many as one fourth of the intraepithelial lymphocytes expressed IFN-gamma. Intraepithelial T lymphocytes in treated, symptom-free celiac patients still had increased IFN-gamma levels compared with controls., Conclusions: In celiac patients, gluten intake seems to cause an overreaction in intraepithelial T lymphocytes, with uncontrolled production of IFN-gamma and IL-10. This may cause both recruitment of intraepithelial lymphocytes and a leaky epithelium, leading to a vicious circle with amplified immune activity and establishment of intestinal lesions.

Background and Aims: A histopathological feature considered indicative of ulcerative colitis (UC) is the so-called basal lymphoid aggregates. Their relevance in the pathogenesis of UC is, however, unknown. We have performed a comprehensive analysis of the immune cells in these aggregates most likely corresponding to the lymphoid follicular hyperplasia also described in other colitides., Methods: Resection specimens of UC and normal colon were analysed by immunomorphometry, immunoflow cytometry, and immunoelectron microscopy, using a large panel of monoclonal antibodies., Results: (1) In all cases of UC, colonic lamina propria contained numerous basal aggregates composed of lymphocytes, follicular dendritic cells, and CD80/B7.1 positive dendritic cells. (2) CD4(+)CD28(-) alphabeta T cells and B cells were the dominant cell types in the aggregates. (3) The aggregates contained a large fraction of cells that are normally associated with the epithelium: that is, gammadelta T cells (11 (7)%) and alpha(E)beta(7)(+) cells (26 (13)%). The gammadelta T cells used Vdelta1 and were CD4(-)CD8(-). Immunoelectron microscopy analysis demonstrated TcR-gammadelta internalisation and surface downregulation, indicating that the gammadelta T cells were activated and engaged in the disease process. (4) One third of cells in the aggregates expressed the antiapoptotic protein bcl-2., Conclusions: Basal lymphoid aggregates in UC colon are a consequence of anomalous lymphoid follicular hyperplasia, characterised by abnormal follicular architecture and unusual cell immunophenotypes. The aggregates increase in size with severity of disease, and contain large numbers of apoptosis resistant cells and activated mucosal gammadelta T cells. The latter probably colonise the aggregates as an immunoregulatory response to stressed lymphocytes or as a substitute for defective T helper cells in B cell activation. gammadelta T cells in the aggregates may be characteristic of UC.

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 human gut epithelium is a unique immunological compartment, containing substantial amounts of intra-epithelial lymphocytes (IEL) with unknown functions. In this study we show that distinct and unusual subpopulations of IEL are present at different levels of human intestine. IEL phenotypes in normal jejunum, ileum and colon were compared using immunoflow cytometry and immunohistochemistry. The expression of mRNA for recombination-activating gene-1 (RAG-1) in IEL from all three levels was compared using reverse-transcription polymerase chain reaction, and the morphology of IEL in situ was determined using immunoelectron microscopy. Surface marker profiles of isolated intestinal epithelial cells at all three levels were also investigated. On average the proportion of TCR gamma delta IEL was comparable in jejunum than ileum and colon and varied in phenotype with gut level. CD4-CD8-TCR alpha beta IEL dominated in colon but were absent in jejunum. CD8+ TCR alpha beta IEL were present at all levels but only in jejunum did they constitute the majority of all IEL. CD4+ TCR alpha beta IEL were present in similar frequencies at all levels of the gut. In general, the majority of IEL had an activated phenotype (CD45RO+, alpha E beta 7+). Furthermore, IEL exhibited phenotypes which are rare in peripheral blood. The thymocyte markers CD1a and CD1c as well as the NK cell marker CD56 were expressed on a fraction of TCR alpha beta and TCR gamma delta IEL. A small population of 'null' cells (CD45+ TCR/CD#-CD20-CD14-CD15- cells) was also present at equal proportions along the gut. Jejunal but not colonic IEL expressed RAG-1 mRNA suggesting that extrathymic T cell maturation occurs in the epithelium of small intestine. RAG-1 was expressed in CD2+TCR/CD3- and CD3+/TCR-IEL. Ultrastructurally, IEL often formed small clusters and intimate contacts with epithelial cells, suggesting cell cooperation within the epithelium. Some IEL had pseudopodium-like extensions penetrating the epithelial basement membrane suggesting transmigration. Epithelial cells in small intestine but not colon expressed heat shock protein 60 and HLA-DR. CD1a, CD1b and CD1c were not expressed on intestinal epithelial cells at any level. The distinct surface marker profiles of IEL and epithelial cells along small and large intestine suggest functional regional specialization and are compatible with the hypothesis that TCR alpha beta IEL participate in immune reactions to lumenal antigens while TCR gamma delta IEL perform surveillance of the epithelium.

A mild purification method has been developed for the isolation of human intraepithelial lymphocytes (IEL) and enterocytes from the same individual. The isolation procedure includes mechanical disruption of the mucosal layer, treatment with reducing agent and sedimentation followed by Percoll gradient centrifugation. Finally, epithelial cells are removed from the IEL fraction using magnetic beads coated with the anti-epithelial antigen monoclonal antibody (mAb) BerEP4. Leucocytes are removed from the enterocyte fraction using magnetic beads coated with mAbs directed against common leucocyte antigen (CD45). Using this procedure IEL and enterocytes have been isolated from apparently normal jejunal, ileal and colonic tissue specimens. Recoveries of IEL were 7 x 10(5), 4 x 10(5) and 1 x 10(5)/cm2 mucosa from jejunum, ileum and colon respectively. 1-2 x 10(6) enterocytes/cm2 mucosa were recovered from small intestine while the corresponding value for colonic biopsies was approximately 2 x 10(5) enterocytes/cm2. The IEL fraction was pure as judged by the low percentages of B cells, macrophages and BerEP4 positive cells (less than 4%) present in the purified fraction. The enterocyte fraction contained less than 2% CD45+ cells. The two cell fractions were viable and expanded in vitro. Enterocytes expanded spontaneously while IEL required initial stimulation with mitogens. The isolation procedure described here will make it possible to study the function of human IEL, interactions between IEL and enterocytes and the role of both cell types in local immunity.

The modulatory effect of human milk proteins from colostrum and late milk on the proliferative response of human T lymphocytes activated by mitogens (OKT3 and leucoagglutinin from Phaseolus vulgaris) and alloantigens was studied. High concentrations (10-100 micrograms/ml) of crude colostral milk proteins had an inhibitory effect on T cell growth while low concentrations (0.1-1 microgram/ml) enhanced T cells growth. In contrast, proteins from late milk did not inhibit T lymphocyte proliferation while the enhancing effect was retained. Colostrum was fractionated by ammonium sulphate precipitation and gel filtration on sepharose 6B. The inhibitory activity was recovered in a protein fraction containing lactoferrin as its major component. Lactoferrin was, however, not responsible for the observed inhibition. On the contrary, lactoferrin in most cases augmented the proliferative response induced by polyclonal activators. The inhibitory activity was found to bind concanavalin A-sepharose suggesting an association with glycoprotein. Inhibitory fractions contained glycoproteins of the following molecular sizes 26, 74/76 (doublet), 84, 145 and 160 kD under reducing conditions. The inhibitory effect appeared to be lymphocyte specific since the active fraction did not inhibit the growth of tissue culture cells (HeLa cells and human fibroblasts) or bacteria. Furthermore, the fraction was not toxic for lymphocytes. The inhibitory colostrum factor may prevent the newborn from overreacting immunologically against the environmental antigens encountered at birth.

Three rabbit antisera (870, 872 and 873) were raised against leucoagglutinin-reactive components from the surface of human T cells. The antibodies reacted with two major glycoproteins of 175 kDa and 105 kDa. None of the antibodies triggered peripheral blood lymphocytes or T cells to proliferation when tested under different culture conditions. All antibodies inhibited the proliferative response to concanavalin A or to allogeneic lymphocytes in mixed lymphocyte culture when whole IgG fractions were used. Complete inhibition of cytotoxic activity was obtained in cell-mediated lympholysis and in natural killer cell cytotoxicity (NK) when fresh peripheral blood lymphocytes were used as effector cells. Weak inhibition was also obtained in NK when mixed lymphocyte culture-activated effector cells were used. The inhibition was stronger, when NK activity was determined against MOLT4 target cells as compared to K562. Whereas F(ab')2 fragments of 873 IgG inhibited cytotoxic T lymphocyte activity completely, Fab fragments of 873 IgG neither inhibited proliferation nor cytotoxic T lymphocyte activity, but gave some inhibition of NK against MOLT4 targets. The results indicate that antibodies against these leucoagglutinin-reactive structures reacted with polypeptides similar to or identical with the human "leukocyte function-associated antigen-1" (LFA-1) considered to be an important mediator of cell-cell interactions and nonspecific adherence.

CD43 (large sialoglycoprotein) is a heavily glycosylated protein expressed on virtually all thymus-derived lymphocytes, on a subpopulation of B cells and on granulocytes. Recently, an anti-CD43 mAb (L10) was shown to induce proliferation in T cells comparable to that induced by anti-CD3. The L10 antibody was reported to react with both sialylated and desialylated CD43. In order to further elucidate the role of CD43 in various T cell functions we have studied the biologic properties of two other mAb (B1B6 and E11B, IgG1) directed against sialic acid-dependent epitopes on CD43. Addition of low amounts of antibody (5 to 10 ng/ml) to freshly isolated T cells or to T cell lines resulted in a rapid clustering of the cells. Fab fragments were also active albeit at a 10-fold higher concentration. Aggregation was dependent on active cell metabolism (inhibited by azide and at low temperatures), on the presence of divalent cations (Mg2+) and was inhibited by antibodies to CD18 but not by antibodies to CD11a (leukocyte function-associated Ag-1 alpha). B1B6 and E11B were poorly mitogenic when added alone in soluble form to PBL or to T cells. However, supernatants from cultures of PBL treated with B1B6 for 2 days contained IL-2 activity. No increase in the number of CD25+ cells was seen during the same period. Exogenously added IL-2 did not synergize with B1B6 or E11B in activation of PBL, whereas proliferation was significantly increased by the addition of the antibodies to activation systems with low endogenous production of IL-2 (PMA or soluble anti-CD3). The anti-CD43 antibodies amplified T cell proliferative responses induced by Con A or leukoagglutinin from Phaseolus vulgaris. F(ab')2 fragments enhanced proliferation significantly better than Fab fragments suggesting that cross-linking of CD43 molecules was an essential features of the amplifying signal. Compared with cultures activated by Con A alone, an increased number of CD25+ cells and of blast cells as well as an increased IL-2 production was observed in cultures activated by B1B6-Con A. The results indicate that regulatory signals, which may function to modify homo- or heterotypic T cell adhesion as well as autocrine production of IL-2, can be transduced through CD43.

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.

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.

Bovine peripheral blood lymphocytes (PBL) were isolated from blood collected from 6 cattle. After treatment with neuraminidase, 40 or 60% of the cells were shown to combine with Helix Pomatia A hemagglutinin (HP) depending whether a direct or indirect fluorescence technique was used. About 20% of the cells were Ig-bearing. With double staining fluorescence technique, it was shown that cells attaching to HP were not Ig-bearing and the reverse. With the aid of HP, covalently bound to Sepharose, Ig-bearing cells could be separated from cell populations attaching to HP. The fraction of cells forming rosettes with sheep erythrocytes was proportional to that of HP attaching cells both before and after fractionation on the HP column. It is therefore concluded that HP is a marker for bovine T-cells, and that this lectin may be used to separate B-cells from T-cells.

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.

Chickens were immunized with highly purified large sialoglycoprotein of human lymphocytes (L-LSGP; gp 150) which was isolated from neuraminidase-treated normal peripheral blood lymphocytes by affinity chromatography to HP-Sepharose and further purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies isolated from plasma and egg yolk were highly specific for desialylated L-LSGP (apparent molecular mass approximately 150 kDa). The antigenic sites recognized by the antibodies are probably located in the peptide moiety of the molecule since antibody binding to lymphocytes was not inhibited by a variety of different sugars or abrogated by absorption on various erythrocytes. In immunofluorescence experiments, greater than or equal to 75% of neuraminidase-treated thymocytes and peripheral blood lymphocytes, virtually all E+ cells and T4+ or T8+ T chronic lymphocytic leukemia (CLL) cells were stained by anti-gp 150. A small fraction (approximately 10%) of thymocytes and a larger fraction (greater than or equal to 30%) of T CLL cells in some patients were stained before neuraminidase treatment. Thymocytes appear to contain considerably lower amounts of a less sialylated form of L-LSGP than peripheral blood lymphocytes. In contrast to blast cells of 5-day concanavalin A or leucoagglutinin cultures (greater than or equal to 90% anti-gp150+) only about 50% of the blast cells generated in 5-day mixed leukocyte cultures were anti-gp150+. The large majority (greater than or equal to 75%) of both the anti-gp150+ and anti-gp150- cells were T3+ and T11+.(ABSTRACT TRUNCATED AT 250 WORDS)

T cells from human peripheral blood was purified by fractionation on columns charged with human immunoglobulin and rabbit anti-human immuno-globulin. When assayed with 125I- or fluorescein isothiocyanate-labeled wheat-germ agglutinin (WGA), a weakly binding and a strongly binding subpopulation could be distinguished. These T-cell subpopulations were fractionated on columns charged with WGA, convalently bound to Sepharose 6MB. The cells responding to the mitogens leukoagglutinin from Phaseolus vulgaris and concanavalin A were enriched in the strongly binding subpopulation (approximately 20% of the T cells) while they were depleted from the weakly binding subpopulation.

Human and mouse lymphocytes were surface-labeled by lactoperoxidase-catalyzed iodination, or by galactose oxidase oxidation followed by reduction with tritiated sodium borohydride. The labeled cells were lysed with Nonidet P-40. Proteins binding to Helix pomatia A hemagglutinin (HP) were isolated by affinity chromatography on HP-Sepharose and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. A major cell surface glycoprotein (apparent mol. wt. 150 000, using reducing conditions) on human lymphocytes was responsible for almost all binding of HP. This protein was present on normal and malignant thymus-derived lymphocytes, e.g. thymocytes, blood T cells and T leukemia cell lines. It was also found on chronic lymphocytic leukemia cells, one null cell leukemia line, one unidentified leukemia line, one lymphoblastoid cell line of B origin and on one stem cell lymphoma line. In contrast, this protein was not found on various B cells at different steps of differentiation, e.g. four B lymphoma lines or one myeloma line. It was also absent from a histiocytic leukemia line. However, two of the four B lymphoma lines and the myeloma line had another HP-binding surface glycoprotein (mol. wt. 200 000) instead of the 150 000 protein. Studies of mouse lymphocytes similarly showed that thymus-derived lymphocytes (normal and malignant) but not normal adult B cells expressed a major HP-binding surface glycoprotein of apparent mol. wt. 130 000 (reducing conditions).

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.

Large sialoglycoprotein of human lymphocytes (L-LSGP) from thymocytes and from peripheral blood lymphocytes (PBL) of normal donor and of B chronic lymphocytic leukemia (CLL) patients was purified by affinity chromatography to Maclura pomifera agglutinin (MPA)-Sepharose followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). L-LSGP from the three different sources was very similar in amino acid composition. It contained a high proportion of acidic and hydroxy amino acids and also significant amounts of cysteine. No reduction in mobility in SDS-PAGE was noted for unreduced L-LSGP. The molecule may already in its native form have an extended conformation containing either free sulfhydryl groups or small S-S loops not affecting mobility in SDS-PAGE. L-LSGP was found to be highly glycosylated, the thymocyte glycoprotein containing somewhat less carbohydrate by weight (44%) than that of PBL (normal PBL 53% and B CLL 52%). This was due primarily to a lower content of sialic acid. The molecules contained mannose, galactose, N-acetyl galactosamine, N-acetylglucosamine and sialic acid in molar ratios 1.0:3.0:1.1:1.2:1.3 (thymocyte L-LSGP), 1.0:3.8:1.2:1.0:1.7 (PBL L-LSGP) and 1.0:3.5:2.2:1.3:2.8 (B CLL L-LSGP). The weak interaction of L-LSGP with lentil lectin, concanavalin A (Con A) and leucoagglutinin (La), its unchanged mobility in SDS-PAGE after tunicamycin treatment and its high amount of hydroxy amino acids suggest that most carbohydrate chains are O-glycosidically linked to the peptide chain. Native as well as Nase-treated L-LSGP show size microheterogeneity. This is probably due to small chemical differences in the L-LSGP molecules from different lymphocyte subsets.

Neuraminidase treatment of human peripheral blood lymphocytes uncovers cell surface receptors that bind purified A hemagglutinin from the snail Helix pomatia. No hemagglutinin was bound to untreated lymphocytes. Binding studies with (125)I-labeled hemagglutinin suggested that the number of receptors on neuraminidase-treated lymphocytes was approximately 1.10(6)/cell. The apparent association constant for hemagglutinin binding to lymphocytes, as calculated from Scatchard's plots, was 5-7.10(8) liters/mol. Immunofluorescent staining with FITC-conjugated hemagglutinin gave positive reactions with approximately 60% of the lymphocytes from normal donors. Positive staining was inversely related to the number of lymphocytes with Fc or complement receptors or with surface immunoglobulin, thus suggesting that See PDF for Structure the lymphocytes with receptors for Helix pomatia A hemagglutinin are T cells. Cell fractionation on columns charged with hemagglutinin indicate that these receptors may be used for separating subpopulations of human peripheral lymphocytes.

Two out of 20 monoclonal antibodies (IgM, kappa), mAb 3192 and mAb K3G, raised against leucoagglutinin-reactive components on human T cells, effectively blocked lymphocyte-mediated cytotoxicity in vitro. No antigenic polypeptide reactive with these antibodies has been identified thus far. However, they have previously been shown to react specifically with certain neutral glycolipids obtained from spleen. Both mAb inhibited the cytotoxicity of natural killer (NK) cells against K562 cells, antibody-dependent cellular cytotoxicity (ADCC) towards antibody-coated bovine erythrocytes and cytotoxic T lymphocyte activity against allogeneic target cells. In both NK and ADCC, preincubation of the lymphocytes with different antibody concentrations resulted in a dose-dependent reduction of cytotoxicity. In contrast, preincubation of the target cells had no effect indicating that the mAb inhibited cytotoxicity at the effector cell level. When studied at the single-cell level, the mAb did not alter the number of lymphocytes forming conjugates with K562 but significantly reduced the frequency of conjugates containing dead target cells. Addition of the mAb to preformed conjugates resulted in a dose-dependent reduction in the proportion of conjugates containing dead target cells. Furthermore, mAb 3192 did not reduce the number of lymphocytes forming rosettes with bovine erythrocytes, indicating that inhibition of ADCC was not due to blocking of the effector cell-target cell interaction mediated by the Fc receptor of the effector cells. Taken together, these results suggest that the mAb inhibited cytotoxicity by interfering with a post-binding step common for the different cytotoxicity systems.

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.