Branched I antigens on leukemia cells enhanced sensitivity against natural killer-cell cytotoxicity through affecting the target-effector interaction.

BACKGROUND: The aberrant glycosylation on proteins and lipids has been implicated in malignant transformations for promoting the tumorigenesis, metastasis, and evasion from the host immunity. The I-branching ß-1,6-N-acetylglucosaminyltransferase, converting the straight i to branched I histo-blood group antigens, reportedly could influence the migration, invasion, and metastasis of solid tumors. STUDY DESIGN AND METHODS: We first chose the highly cytotoxic natural killer (NK)-92MI cells as effector against leukemia for this cell line has been used in several clinical trials. Fluorescence-activated cell sorting and nonradioactive cytotoxicity assay were performed to reexamine the role of NK-activating receptors, their corresponding ligands, and the tumor-associated carbohydrate antigens in this NK-92MI-leukemia in vitro system. The I role on cytotoxic mechanism was further studied especially on the effector-target interactions by cytotoxic analysis and conjugate formation assay. RESULTS: We showed that expression levels of leukemia surface ligands for NK-activating receptors did not positively reflect susceptibility to NK-92MI. Instead, the expression of I antigen on the leukemia cells was found important in mediating the susceptibility to NK targeting by affecting the interaction with effector cells. Furthermore, susceptibility was shown to dramatically increase while overexpressing branched I antigens on the I- cells. By both conjugate and cytotoxicity assay, we revealed that the presence of I antigen on leukemia cells enhanced the interaction with NK-92MI cells, increasing susceptibility to cell-mediated lysis. CONCLUSION: In our system, branched I antigens on the leukemia were involved in the immunosurveillance mediated by NK cells specifically through affecting the effector-target interaction.

Molecular genetics of the blood group I system and the regulation of I antigen expression during erythropoiesis and granulopoiesis.

PURPOSE OF REVIEW: The molecular genetics of the blood group I system and the regulation mechanism for I antigen expression in postnatal red blood cells are intriguing. This review summarizes their elucidation and recent findings. RECENT FINDINGS: Accumulating data from the molecular analysis of individuals with the adult i phenotype supports the proposed molecular genetic mechanism for the partial association of the adult i phenotype with congenital cataracts. Recent investigations have shown that the regulation of I antigen formation during erythropoiesis is determined by transcription factor CCAAT/enhancer binding protein-α (C/EBPα) and the phosphorylation status of C/EBPα Ser-21 residue. SUMMARY: The human I locus is organized such that it has an uncommon genetic architecture and expresses three different I transcript forms. The results obtained from molecular analysis of two adult i groups, with and without congenital cataracts, demonstrate that the molecular background accounts for the partial association between these two traits and suggest that an I gene defect may lead directly to the development of congenital cataracts. Analysis of the regulation for I antigen expression shows that the regulation during erythropoiesis and granulopoiesis share a common mechanism, with dephosphorylation of the Ser-21 residue on C/EBPα playing the critical role.

An unusual case of an acute hemolytic transfusion reaction caused by an autoanti-I.

In general, naturally occurring cold autoagglutinins react optimally at low temperatures. We describe a young child who experienced an acute hemolytic transfusion reaction by an unusual autoanti-I. The IgM autoanti-I was detected at 4°C (titer 256) and also reacted at 30°C. This case highlights the potential hazard of transfusing units of blood immediately upon removal from the blood refrigerator, especially into neonates and children of small stature.

Clinical evaluation for lymphoproliferative disease prompted by finding of IgM warm autoanti-IT in two cases.

Anti-IT is an unusual specificity originally described as a naturally occurring cold agglutinin. The antibody reacts strongly with cord RBCs, weakly with adult I RBCs, and most weakly with the rare adult i RBCs. IgG anti-IT in patients with hemolytic anemia has been associated with Hodgkin's lymphoma. Difficulties in blood grouping tests and the presence of a warm reactive agglutinin in samples from two patients with hemolytic anemia led to further serologic studies and the identification of anti-IT. In both cases, the anti-IT was a rarely encountered IgM warm reactive agglutinin; in one case, the IgG component was also anti-IT, whereas in the second case the IgG antibody was broadly reactive. The unusual serologic finding of anti-IT prompted further clinical evaluation for lymphoproliferative disease in these two patients.

A 13-year-old girl with cold agglutinin syndrome caused by anti-i.

SUMMARY: A 13-year-old girl with cold agglutinin syndrome caused by anti-i was serologically positive for Epstein-Barr virus. The anti-i had a high titer at 4 degrees C and high thermal amplitude (reacting up to 37 degrees C with both cord i RBCs and the patient's autologous RBCs). The patient's hemoglobin dropped to 48 g/L. The age of the patient, the severity of the hemolysis, and the antibody specificity were unusual features of cold agglutinin syndrome. Transfusions with adult (I) red blood cells were effective.

Anti-D and anti-i activities are inseparable in V4-34-encoded monoclonal anti-D: the same framework 1 residues are required for both reactivities.

BACKGROUND: The heavy-chain V4-34 germline gene segment is mandatory for pathologic cold-reacting autoantibodies with anti-I/i specificity (cold agglutinins) and is also preferentially used by monoclonal immunoglobulin M alloantibodies against D and other Rh antigens. The use of the V4-34 segment by monoclonal anti-D has previously been shown to also confer anti-I/i reactivity (cold agglutinin activity), which has implications for the use of such antibodies for Rh blood typing. V4-34 framework 1 (FR1) sequence is believed to be critical for cold agglutinin activity of cold agglutinins. STUDY DESIGN AND METHODS: The aim of this investigation was to use site-directed mutagenesis of a recombinant V4-34-encoded anti-D to determine the contribution of V4-34 FR1 sequence to anti-D activity and whether mutational modifications in the FR1 region could separately alter anti-D and anti-i activities. RESULTS: The results show that amino acid changes in V4-34 FR1 at W7, A23, and Y25 have a profound effect on anti-D activity as well as on anti-i activity. It was not possible to substantially reduce or remove anti-i activity without reducing anti-D activity to a comparable extent. CONCLUSIONS: The same nonpolar hydrophobic amino acids in FR1 are critical for maintaining both anti-D and anti-i activity. It is proposed that these residues influence the conformation of the antigen-binding site.

A case of recurrent paroxysmal cold hemoglobinuria with the different temperature thresholds of Donath-Landsteiner antibodies.

In this article, we first report a case of recurrent paroxysmal cold hemoglobinuria with serologic confirmation. On 2 occasions, the Donath-Landsteiner (DL) antibodies belonged to an IgM subclass and showed neither anti-P nor anti-I specificity. Furthermore, it is very interesting that the temperature thresholds of DL antibodies were different on each occasion. Although acute paroxysmal cold hemoglobinuria is considered to be self-limited and transient, we should be careful of its possible recurrence. DL tests must be repeated after the complete recovery from the first episode, with careful attention to several possible causes of false-negative DL tests.

[Autoantibodies, tolerance and autoimmunity]. / Autoanticorps, tolérance et auto-immunité.

In 1900, the group from Metchnikoff suggested the concept of autoimmunization by demonstrating the presence of autoantibodies in normal conditions; which was opposed to the concept of horror autotoxicus raised by Ehrlich. Landsteiner's description of the transfusion compatibility rules and 50 year-later work from Burnett's and Medawar's groups lead to the clonal deletion theory as a general explanation of tolerance and autoimmunity. However, more recent work succeeded demonstrating that autoreactive B cells constitute a substantial part of the B-cell repertoire and that this autoreactive repertoire secretes the so-called natural autoantibodies (NAA) characterized by their broad reactivity mainly directed against very well conserved public epitopes. They fulfill the definition of an autoantibody since they are self-reactive, but they are not self-specific. As yet, NAA directed against determinants of polymorphism have not been reported. The presence of this repertoire in normal conditions challenges the clonal deletion theory as a unique explanation for self-tolerance. However, if we take into account that this autoreactive B-cell repertoire is not self-specific, this contradiction may not be a real one opposition. Indeed, the Lansteiner's rule that a subject belonging to group A will never produce anti-A antibodies and will always produce natural antibodies against the B-cell group, could never be challenged. Clonal deletion is probably accounting for this phenomenum. However, the serum of healthy adult individuals frequently exhibits low titers of anti-I antibodies, which is a precursor molecule of AB0 antigen system. The mechanism accounting for deletion of B cells directed against critical determinants like antigens A and B in the red blood cell system and allowing the production of autoantibodies against I remain elusive.

Lectins as markers for blood grouping.

Lectins are unique proteins of varying biological importance. They are characterized by specific binding to carbohydrate residues, whether monosaccharides, disaccharides or polysaccharides. The sugar heads on the surface of the erythrocyte specify the different blood groups. Lectins, as an antigenic determinant of blood group, have come to be an important tool in the identification of different blood groups. A handful of lectins may be considered excellent reagents for anti-A, anti-B, anti-N etc, but the anti-A and anti-M are not yet regarded as commercially suitable antisera. Lectin from Vicia cracca has been proved to be a good anti-A, lectin from Dolichus biflorus can be used as anti-A1, and lectin from Griffonia simplicifolia as anti-B. Lectin from Vicia graminea is said to be a good typing reagent as Anti-N. On the other hand, the lectins involved in polyagglutination are absolutely essential as the reagent of choice and these cannot as yet be replaced by antibodies of any kind. Erythrocytes with exposed cryptantigens are significantly more sensitive to agglutination by certain lectins than by polyclonal antibodies. Peanut agglutinin (PNA), Polybrene, and Glycine max lectins are frequently used for the identification of different cryptantigens. The application of lectins as an anti-B reagent has proven to be as useful as human polyclonal or mouse monoclonal antibodies. Besides their specificity, lectins are excellent reagents because of their lower cost and indigenous production. The importance of various lectins used as markers for blood grouping is discussed.

A comparison of the Aplysia lectin anti-I specificity with human anti-I and several other I-detecting lectins.

BACKGROUND: Lectins displaying blood group specificity are important for blood group typing and antigen recognition. Their use in blood banks is especially widespread in situations where there is a shortage of specific antisera. This report describes the efficiency of Aplysia gonad lectin as a reliable reagent for the detection of I antigen, which is common on adult human cells but reduced in fetal, newborn, and rare adult red cells. STUDY DESIGN AND METHODS: The selective hemagglutinating activity of the Aplysia lectin was compared with that of human anti-I and several I-reactive lectins, including two plant lectins, one galactophilic microbial lectin, and bovine spleen galectin. RESULTS: The comparison has revealed that Aplysia gonad lectin, like human anti-I, strongly agglutinates and adsorbs to adult I-positive red cells, differentiating between them and fetal or rare I-negative adult red cells (although with less of a difference). In contrast to the plant and microbial lectins examined, its I-affinity does not depend on the presence of ABH or P system antigens and it clearly detects higher I antigen expression in Oh red cells. The hemagglutinating activity of Aplysia lectin as that of all the I-detecting proteins is enhanced at 4 degrees C, but unlike the human anti-I Aplysia lectin-induced hemagglutination is stable at room temperature. CONCLUSIONS: The Aplysia lectin is a reliable anti-I reagent, which strongly agglutinates I-positive adult human red cells irrespective of their ABH or P system antigens. This lectin is usable at room temperature.

A comparison of different density gradients and antibodies for enrichment of fetal erythroblasts by MACS.

The enrichment of fetal cells, in particular fetal erythroblasts from the blood of pregnant women offers a promising non-invasive alternative for prenatal diagnosis. The purpose of this study was to compare the retrieval of erythroblasts by different density gradients and different antibodies against erythroid surface antigens, in both a model test system and in blood samples of pregnant women. We enriched erythroblasts from artificial mixtures of cord and adult blood (1:50) and from 16 ml of peripheral blood from pregnant women at a mean gestational age of 14+2 weeks. The yield of erythroblasts was calculated and compared using Wilcoxon's matched-pairs signed-rank test. In the artificial mixture most erythroblasts were retrieved using the heaviest density gradient (specific density of 1119) followed by antibody-labelled magnetic cell sorting (MACS). With anti-GPA the yield of erythroblasts from the artificial mixture was highest (9362.5 erythroblasts) compared with anti-CD36 (5164.3), anti-i (3455.2), anti-CD71 (3055.8) and HAE9 (2364.2). The difference between anti-GPA and anti-CD71, HAE9 and anti-i was significant (p=0.0277). The enrichment of erythroblasts from peripheral blood of pregnant women showed similar results. The yield of erythroblasts using anti-GPA was the highest. These results enable us to simplify our enrichment protocols to a single density gradient of 1119 specific density followed by MACS, with anti-GPA.

Cold-induced augmentation of I blood group antigen interactions with galactophilic lectins.

The I antigen appears on human cells in the postnatal period, by addition of N-acetyllactosamine (beta 1-6) branching to the fetal i antigen structure, which is specified by linear oligo N-acetyllactosamine (beta 1-3) chain. Concurrently with the I antigen appearance on adult human erythrocytes most human sera exhibit low levels of anti-I agglutinins. These antibodies induce hemagglutination mainly at low temperatures (4 degrees C) and scantly at body temperature. Therefore they were named "cold agglutinins". We have used these antibodies and several hemagglutinating galactophilic animal, plant, and microbial lectins that also react with the I antigen, to study whether the cold-favored agglutination of the I antigen-bearing cells is a peculiar property of the anti-I antibodies or a special trait of that antigen. It has been found that the interactions of all of the examined lectins, irrespective of their source, with the adult human erythrocytes significantly increased at 4 degrees C, in contrast to those of the same cells with diverse I-insensitive antibodies and lectins, which were significantly higher at room temperature.

Pancreatic tumour marker anti-mucin antibody CAM 17.1 reacts with a sialyl blood group antigen, probably I, which is expressed throughout the human gastrointestinal tract.

CAM 17.1 is an antimucin monoclonal antibody which has recently been proven valuable as a reagent for serological diagnosis of pancreatic cancer. A series of studies have been performed to characterise its epitope. First it was screened immunohistochemically against a wide range of formalin-fixed normal and neoplastic human tissues and showed widespread binding to mucin throughout the gastro-intestinal tract, in both normal and malignant tissues. In pancreas, strong intracellular staining of acinar and ductal cells was found in normal tissue and in carcinoma cells in tumours. Normal stomach showed only weak staining (n = 6), but gastritis with metaplasia showed strong staining (n = 4). Staining of colonic mucosa from patients of known Lewis phenotype showed Le(a+b-) (7/8) and Le(a-b+) (4/6) samples to be positive, but not Le(a-b-) (0/3) samples. CAM 17. 1 agglutinated all donor erythrocytes tested at 4 degreesC regardless of blood group, whereas cord blood red cells were not agglutinated. Since I antigen is the only antigen known to be present on all adult red blood cells but absent from cord blood, this suggests probable involvement of this antigen in the binding site. The agglutination was abolished by sialidase treatment of the red cells and immunoblotting with slot-blotted mucin showed that binding was both acid and sialidase sensitive indicating the involvement of sialic acid in the binding site. These studies show that CAM 17.1 binds to a sialic-acid-containing determinant of mucin, probably sialyl-I, which epitope shows wide distribution throughout the gastro-intestinal tract.

Cold agglutinin disease by autoanti-i blood type antibody associated with B cell chronic lymphocytic leukemia.

Cold agglutinin (CA) disease is a rare complication of B cell chronic lymphocytic leukemia (CLL). We report a case of CA disease by autoanti-i blood type antibody associated with CD5-negative CLL. The specificity of cold agglutinin in the sera and eluates from red blood cells were analyzed by reactivity against panels of red blood cells and also hemagglutinin inhibition assay using lactonorhexaosylceramide, and were determined to be an anti-i blood group antibody of the monoclonal IgM kappa isotype. PCR analysis of immunoglobulin heavy-chain genes from bone marrow-derived genomic DNA showed CLL cells utilizing DP-54 as a VH gene, differing from the reported VH genes used for cold agglutinin with anti-I/i specificity. The anti-i antibody in this patient might be secondarily produced by residual B cells due to an immunodysregulatory state, and not from CLL cells.

Inverse correlation between IgG-antihinge region and antierythrocyte autoantibody in chronic benign and malignant cold agglutination.

Previous reports provided evidence of an immunosuppressive role of natural anti-F(ab')2 antibodies. If suppressive anti-F(ab')2 antibodies also regulated the autoantibody production in cold agglutination, one would expect high titers of anti-F(ab')2 to be associated with low titers of cold agglutinins. Indeed, our previous studies revealed an inverse correlation between IgG-anti-F(ab')2 and cold agglutinins. Many previous experiments focused on anti-F(ab')2 of an antiidiotypic nature. Recent epitope mapping showed that anti-F(ab')2 of healthy persons is not an antiidiotype but recognizes a hinge region sequence. We attempted to answer the question whether this IgG-antihinge antibody is responsible for the previously described association between anti-F(ab')2 and cold agglutinins. IgG-antihinge and IgG-anti-F(ab')2 antibody was determined and statistically analyzed in the serum of 334 patients with cold agglutination. Our experiments revealed a strong correlation between the concentrations of antihinge and the previously described anti-F(ab')2 antibody. The anti-F(ab')2 activity was competitively inhibited by a synthetic hinge peptide. Moreover, patients with high antihinge titers had low cold agglutinin titers, and vice versa. A stratification according to cold agglutinin specificity and disease etiology showed that the inverse correlation is present only in anti-I and anti-i patients suffering from monoclonal B-lymphocyte proliferation. In conclusion, our results confirm the correlation previously described for anti-F(ab')2 antibody and antierythrocyte autoantibody and define for the first time an association between an idiotype-independent anti-IgG autoantibody and cold agglutinin.

Cold agglutinin activity is common among human monoclonal IgM Rh system antibodies using the V4-34 heavy chain variable gene segment.

BACKGROUND: The V4-34 gene segment is commonly used by human monoclonal IgM alloantibodies against blood group antigens and by cold-reactive red cell autoantibodies with anti-I or anti-i specificity. This study was conducted to determine whether cold agglutinin activity is found among the V4-34-encoded alloantibodies. STUDY DESIGN AND METHODS: Fifty-four human IgM monoclonal antibodies (MoAbs) against Rh system antigens were tested for cold agglutinin activity against red cells lacking the relevant Rh system antigen and for reactivity with tissue I and/or i antigens using immunohistochemistry. The findings were correlated with the utilization of the V4-34 segment as determined in an enzyme-linked immunosorbent assay with an antibody (9G4) that is specific for this gene product and were also correlated with other serologic properties. RESULTS: Of the MoAbs, 59 percent were 9G4-positive. Of the 9G4-positive subset, 16 and 44 percent agglutinated native adult (express I) and cord (express i) cells, respectively, at 4 degrees C; these levels rose to 84 and 94 percent, respectively, with the use of papain-treated cells. The red cell antigens recognized at 4 degrees C were cleaved by endo-beta-galactosidase, which is consistent with their being I and i. Of the 9G4-positive subset, 53 percent bound to tissue i antigen. These reactivities were not found among 9G4-negative MoAbs. Endo-beta-galactosidase treatment of red cells enhanced Rh system antibody agglutination by 9G4-negative MoAbs. CONCLUSION: Anti-I/i reactivity is common among IgM Rh system MoAbs and is shown only by the V4-34-encoded subset. This finding has implications for the use of MoAbs for Rh system typing of blood.

Carbohydrate recognition by Mycoplasma pneumoniae and pathologic consequences.

Mycoplasma pneumoniae infection in the human is often followed by a transient autoimmune hemolytic disorder characterized by high titer autoantibodies to a carbohydrate antigen, the I antigen. Because the major host cell receptor for the Mycoplasma is the sialylated form of this antigen, it is likely that the immunologic disorder is initiated by the microbe-saccharide interaction. Here we review briefly knowledge on the autoantibodies and the structures and distribution of the saccharide antigens and receptors. We discuss possible mechanisms for the triggering of autoantibody production and consider ways in which perturbation of various glycoprotein carriers of the carbohydrate ligands may elicit a variety of pathobiologic responses. We conclude by highlighting ideas on further molecular dissections of the elements of the microbe-host interaction.

The Epstein-Barr virus (EBV) alters the B cell glycolipid which is recognized by the human monoclonal antibody to i-blood group antigen.

To analyze the differentiation-related glycolipids, we have recently developed human monoclonal anti-i antibodies (mAbs) using a combination of EBV-transformation and bovine i-active glycolipid (NeuAc alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc beta 1-->1Cer)-containing liposome immune lysis assay (LILA). Using complement cytolysis with these mAbs, we found the occurrence of a surface antigen (Ag) in the EBV-negative but not in the EBV-positive cell lines. The fresh EBV infection reveals that the suppressed expression of the antigen was a result of the EBV infection. The Ag recognized with these mAbs appeared to have a very low density on the B cell lines. Unexpectedly, thin layer chromatogram (TLC)-immunostaining using the mAbs revealed that the major immunoreactive substance in the EBV-negative B cell lines was an extremely minor glycolipid that was distinct from the i-active glycolipid however, this was not the case in the EBV-positive B cell lines.