P. R. Desai

Human blood-group MN and precursor specificities: structural and biological aspects

The human blood-group MM and NN antigens carry 2 to 4 immunodominant groupings per repeating subunit and differ only by one sialic acid residue per immunodominant group. This residue covers in the MM antigen the beta-D-galactopyranosyl group that is terminal in the N immunodominant structure and that, together with a terminal alpha-linked N-acetylneuraminic acid residue, is responsible for N specificity. M specificity was readily converted into N specificity by mild acid treatment. N structure is the immediate biochemical precursor of M structure, and M and N antigenic specificities are not determined by two allelic genes as believed hitherto. The NN antigen was inactivated by beta-D-galactosidase as well as by removal of N-acetylneuraminic acid. Some of the reactivities of the NN antigen, lost upon beta-D-galactosidase treatment, reappeared on subsequent partial N-acetylneuraminic acid removal. The structure uncovered by complete sialic acid depletion of MN antigens is the Thomsen-Friedenreich T antigen, the specificity of which is determined by beta-D-galactopyranosyl groups. Beta-D-Galactosidase treatment transformed the T antigen into one possessing Tnactivity. The significance of blood-group MN active substances extends to human breast cancer, where MN antigens were found in benign and malignant glands, but some of their precursors in cancerous tissue only.

Common precursors of human blood group MN specificities

Abstract Human blood group MM and NN specific structures have the same precursors. Complete sialic acid removal produced the Thomsen-Friedenreich T antigen which was transformed into Tn antigen by E. coli β- D -galactosidase on red cells as well as on isolated T antigen. MN antigens and their precursors are most clearly defined by isologous human antisera.

Breast cancer patient's cell-mediated immune response to thomsen-friedenreich (T) antigen

Thomsen‐Friedenreich (T) antigenic specificity as determined with human serum anti‐T was found in reactive form in breast adenocarcinomata but not in healthy and generally not in benign breast tissues. T‐antigenic specificity was demonstrable in all metastatic breast carcinoma lesions. T specificity was also present in adeno‐ and squamous cell carcinomata from other organs; it was not found in the four melanomata, one glioblastoma, and seven benign non‐breast tumors. Breast carcinoma patients but not healthy people showed cellular immunity to T antigen in vivo and in vitro. Most striking was the delayed‐type cutaneous hypersensitivity reaction that was positive in over 85% of the ductal breast carcinoma patients tested, negative in over 94% benign breast disease patients, and in all presumably healthy individuals investigated. T antigen is readily available from healthy human red blood cells in uncontaminated form, and free of HL‐A and Australia antigens.

Tn, a carcinoma-associated antigen, reacts with anti-Tn of normal human sera

Tn antigen is the immediate precursor of the carcinoma (CA)‐associated T antigen; both are masked in non‐CA tissues. Tn antigen was detected by absorption of human anti‐Tn antibody in 46 of 50 primary breast CAs and in all 6 metastases originating from Tn‐positive primary CAs. Thirteen of 25 (52%) anaplastic CAs, but only 2 of 15 (13%) well differentiated CAs had more Tn than T; 1 anaplastic CA had neither antigen. Eighteen of 20 benign breast lesions had no Tn; the 2 positive lesions were premalignant. All 19 breast CAs, studied immunohistochemically, reacted strongly with human polyclonal anti‐Tn; benign or normal glandular tissues had minimal or no reactivity. Among live cancer cell lines, the most malignant sublines had more Tn than T on their cell surfaces. Preliminary studies with rodent monoclonal anti‐Tn and anti‐T antibodies gave immunohistochemical reactivity patterns similar to those of the polyclonal antibodies, but the former were less sensitive in absorption tests. Tn is a CA marker that promises to be useful in tumor detection.

Precursors of the blood group MN antigens as human carcinoma-associated antigens.

About 50 years ago, Hiibener, Thomsen and Friedenreich noted that in vitro bacterial infection of human blood may render erythrocytes panagglutinable by one’s own and all human sera, except those of infants, without visible change of the erythrocytes.2n.113 Later it was shown that red blood cells after exposure to influenza viruses became agglutinable.9 This acquired property has come to be known as the (Hubenerl-Thomsen-Friedenreich phenomenon and may cause errors in blood

Blood group Tn-active macromolecules from human carcinomas and erythrocytes: Characterization of and specific reactivity with mono- and poly-clonal anti-Tn antibodies induced by various immunogens☆

In contrast to healthy and noncarcinoma-diseased tissues, greater than 80% of all carcinomas (CAs) tested express immunoreactive O-(2-acetamido-2-deoxy-alpha-D-galacto-pyranosyl)-(1----3)-serine/threon ine [alpha-D-GalpNAc-(1----3)-Ser/Thr] in their glycoproteins. CA cells shed, into the tumors environment, Tn, which is involved in cancer pathogenesis as adhesion molecule and as autoimmunogen. An increase in density of Tn on primary CA frequently parallels augmented CA aggressiveness. Tn-Active glycoproteins of culture-grown human breast CA DU 4475 cells were isolated from cytoplasm and from spent growth medium, and erythrocyte (RBC) Tn antigen was prepared by (1----3)-beta-D-galactosidase treatment of isolated human O RBC MN glycoprotein-derived Thomsen-Friedenreich (T) antigen. Immunochemical, serological, physical, and chemical analyses showed close resemblance of CA- and RBC-derived Tn antigens. The preponderant carbohydrate in both Tn glycoproteins is the alpha-D-GalpNAc residue, and the antigens have a qualitatively and quantitatively similar amino acid composition. Highly specific rodent monoclonal (Mo) anti-Tn antibodies (Abs) were elicited with Tn RBC and normal O RBC-derived Tn antigen, and compared with CA-anti-Tn MoAbs unwittingly evoked by others. A sensitive enzyme immunoassay (EIA) with Tn antigen as solid phase was developed. In this system, highly purified, naturally occurring anti-Tn antibodies, which all humans possess, were more sensitive in quantitating breast CA Tn structures than the anti-Tn MoAbs induced by Tn RBCs, and by RBC- and CA-derived Tn-active antigens. The sensitivity of anti-Tn MoAbs was higher in detecting RBC-Tn.

Patients' immune response to breast and lung carcinoma-associated thomsen-friedenreich (T) specificity

SummaryWe report here sensitive and specific measurement of immune responses of patients with certain kinds of carcinoma toward the physically and chemically well defined T antigen isolated from healthy human erythrocytes. Over 90% of adenocarcinoma tissues tested possess T-specific immunoreactive structures as determined withhuman antisera, in contrast to healthy tissues and benign lesions. Adenocarcinoma patients recognize the carcinoma-associated T antigen as foreign. Delayed-type skin hypersensitivity reaction to T antigen (DTHR-T) was positive in all 25 lung adenocarcinoma patients tested, in 88% of 101 patients with ductal, in 43% of 30 patients with lobular or tubular breast carcinoma and in 9/9 patients with adenocarcinoma of body cavities. Patients of all Stages reacted positively. All 7 patients with small cell lung carcinoma and 3/5 with malignant melanoma had a positive DTHR-T. None of 17 patients with malignant brain tumors, leukemia or Hodgkins disease, sarcoma or thyroid carcinoma reacted. The DTHR-T was specific in that all 77 healthy persons and 48/49 with other diseases, including 23/24 with non-cancer lung disease were negative; one patient with organizing interstitial pneumonitis was positive. This points to a possible source of false positive reactions. 91% of 149 patients with histologically benign breast disease had a negative DTHR-T; the histology of some of the positive ones was reexamined, 2 proved to have carcinoma in situ.nIn vitro leukocyte migration inhibition and scoring of anti-T hemagglutinin titer using the T-anti-T system diagnosed many adenocarcinomata correctly, including 4 whose histology, while turning positive later, was negative at the time. However, these tests were generally less sensitive and specific than the DTHR-T.This system may also be of screening and monitoring value. Surgical removal of primary carcinoma led to a rebound of anti-T in breast carcinoma patients and its renewed decrease in some, prior to clinical recurrence of cancer. Also, the DTHR-T turned from positive to negative in some Stages I and II breast-and T1–2 N0–1 M0 lung adenocarcinoma patients who had no demonstrable relapse during the ensuing observation period.ZusammenfassungWir berichten hier über sensitive und spezifische Bestimmung der Immunatwort von Patienten mit Brust- und Lungenadenokarzinom gegen das physikalisch und chemische definierte T Antigen, isoliert von menschlichen Erythrozyten. Über 90% der untersuchten Adenokarzinomgewebe besaßen T-spezifische immunreactive Strukturen, gemessen mithuman-anti-T Seren, im Gegensatz zu gesunden und gutartig veränderten Geweben. Bei Adenokarzinomträgern wird das karzinom-assoziierte T Antigen vom Organismus als fremd erkannt. Die verzögerte Hautüberempfindlichkeitsreaktion gegen T Antigen (DTHR-T) war in allen 25 Patienten mit Lungenadenokarzinom positiv, in 88% von 101 Patienten mit ductulärem, in 43% von 30 mit lobulärem oder tubulärem Mammakarzinom sowie in 9/9 Patienten mit Körperhöhlen-Adenokarzinomen. Alle 7 Patienten mit kleinzelligem Lungenkarzinom und 3/5 mit malignem Melanom reagierten positiv. Die DTHR-T war negativ in allen 17 Patienten mit malignen Hirntumoren, Leukämien, Hodgkin, Sarkom und Schilddrüsenkrebs. Die DTHR-T war spezifisch: alle 77 gesunden und 48/49 Personen mit nichtkrebsigen Erkrankungen einschließlich 23/24 Lungenpatienten hatten eine negative DTHR-T; ein Patient mit organisierender interstitieller Pneumonitis reagierte positiv; dies deutet auf eine mögliche Fehlerquelle hin. 91% von 149 Patienten, histologisch als gutartige Brusterkrankung diagnostiziert, hatten eine negative DTHR-T; die Histologie einiger positiv reagierender Patienten wurde reexaminiert und erwies sich in 2 Fällen als in situ Karzinom.In vitro „Leukocyte migration inhibition“ und „scoring“ der anti-T Agglutinine ergaben eine korrekte Diagnose bei vielen Adenokarzinomen einschließlich 4 Fällen, die zur Zeit unserer positiven Resultate eine negative Histologie hatten, bei denen spätere Biopsien aber positiv ausfielen. Im allgemeinen waren die in vitro Teste weniger sensitiv und spezifisch als die DTHR-T.Das T-anti-T System könnte sich auch zum „screening“ und „monitoring“ eignen. Chirurgische Entfernung des Primärkarzinoms führte zu einem „rebound“ des anti-T Titers in Brustkrebspatienten, erneuter Abfall wurde in einigen Fällen vor klinischem Rückfall beobachtet. Positive DTHR-T wurde wieder negativ in mehreren Fällen von Brustkrebs Stage I und II bzw. Lungenadenokarzinom T1–2 N0–1 M0; diese Patienten hatten in der folgenden Beobachtungszeit keinen klinischen Rückfall.

Human blood groups M-, N-, T-, and Tn-specific substances in lipidic extracts of line 10 hepatocarcinoma of Strain 2 guinea pigs

Live line 10 (L-10) hepatocarcinoma cells of Strain 2 guinea pigs carry immunoreactive human blood group T (Thomsen-Friedenreich)- and Tn-specificities on their surface as determined with human antisera. Disintegrated L-10 cells show in addition M and N activities; this indicates their presence within the tumor cells. Blood groups S and D(Rh0) activities were not demonstrable. M and N but not Tn and T specificities were also found in normal submaxillary and sublingual glands, kidney, and liver of Strain 2 guinea pigs. After extraction of L-10 carcinoma cells with n-butanol-water, all activities resided in the organic phase. Extraction of the organic phase by Sevags deproteinization procedure yielded nearly eight times as much active material (by weight) in the chloroform layer as in the aqueous layer. Depending on the antisera used, M and N activities of the extracts amounted to 10 to >100% of authentic red cell M and N glycoproteins, Tn activity to ∼75%, and T activity to between 5 and 10% of that of human red blood cell-derived T antigen. The active extracts had <0.75% protein and <3.50% ash. We report two novel findings: (a) the occurrence of M and N specificities in nonprimates, and (b) the association of these specificities with lipidic substances.

Cellular immunity towards Thomsen-Friedenreich antigen in breast-carcinoma patients.

Many women who suffer from breast carcinoma, both at the time of surgery as well as several months later, show a highly significant humoral response to ThomsenFriedenreich (T) antigen when compared with non-carcinomatous control individuals including patients with benign breast disease [1, 2]. We have now found that post-operative breast-carcinoma patients show in vivo and in vitro cellular immune response towards T antigen while presumably healthy individuals do not. T antigen was prepared from isolated human 0,MN red cell antigens and assessed for lack of pyrogenicity as described previously [2, 3]. Patients from Evanston and St. Francis Hospitals, Evanston, IL, who were without therapy for at least 25 days were studied. Blood was collected and white blood cells separated by a procedure similar to that of Clausen [4]. The washed white cells were suspended in Hanks balanced salt solution (HBSS) at a concentration of ca. 1.5 x 108 polymorphonuclear neutrophils (PMN)/ml and incubated for 40 min at 37 ~ in HBSS alone or HBSS containing varying concentrations of T antigen with agitation in a water-saturated atmosphere containing 3% CO2. Agarose medium (1% final concentration) prepared as by Clausen [4] was poured into plastic dishes, 8 equidistant holes were made and 1.5x l0 6 PMNs were added per well. All experiments were set up in triplicate; migration distance of leukocytes was measured with a micrometer after 18 h incubation and migration area was computed. Inhibition of migration was expressed as migration index (M.I.), i.e. the ratio of the average area of migration of leukocytes in presence of T antigen and that in its absence from the same person on the same plate. Identically prepared leukocytes of apparently healthy individuals were included as controls on each plate. In vivo recall delayed hypersensitivity was measured by one i.d. injection of 3 mg T antigen in the arm of 19 breast-carcinoma patients, 2 months to 4 years after surgery. Interpretation of delayed reactions 24-30 h after injection was modified after Nemoto et al. [5] in that a score of 5 was given to averaged perpendicularly measured diameters of 45-54 mm of erythema and a score of 6 to those which were > 54. Three patients had stage IV carcinoma (international nomenclature), two stage III, seven stage II, and nine stage I. Sixteen of these patients suffered from ductal carcinoma without and with additional features, one from invasive and two from in situ lobular carcinoma. Patients who had been operated for stages III and IV breast carcinoma all had recall delayed hypersensitivity reactions with scores between 3 and 5; those who were stage II all scored between 2 and 6. Of the nine patients with stage I breast carcinoma four showed strongly positive scores of 5 and 6, and three had scores of 1 to 2. The remaining two patients did not react; both had non-invasive in situ carcinoma, one intraductal, the other lobular. In contrast 13 healthy persons showed no delayed hypersensitivity and only one of four patients with fibroadenoma-fibrocystic disease (clinically and biopsy) had a score of 1. The in vitro specific cellular immunity of patients with breast carcinoma to T antigen was measured by leukocyte-migration inhibition. T antigen up to a concentration of at least 4,000 pg/ml was not toxic to leukocytes of healthy persons as evidenced by their viability and area of migration. 2he average M.I. at 1,000 pg T antigen/ml was 1.00 for the twenty-seven healthy individuals tested and at 500 gg/ml it was 0.99. At 500 p.g/ml T antigen two of four stage IV breast-carcinoma patients had an M.I. of <0.80 and two showed no inhibition of leukocyte migration. Among four stage III patients two showed an M.I. of <0.90 and two had no response, while of sixteen patients with stage II breast carcinoma seven showed an M.I. of <0.85; among fifteen patients with stage I disease four had an M.I. of <0.90. The M.I. of < 0.90 in 38.4% of breast carcinoma patients shows clearly an inhibition of leukocyte migration by T antigen which was most pronounced in stage II to IV patients. Of 10 patients with benign breast disease only one had an M.I. of <0.90. She suffered from severe bilateral fibrocystic breast disease with metaplasia and calcification. Among twenty-seven presumably healthy individuals an M.I. of <0.90 was found only in one individual, a Vietnam war veteran with numerous vaccinations and antimalarial treatment. We have shown earlier that many gram-negative bacteria possess in vitro T activity [6].

Comparison by leukocyte adherence inhibition of human immune response to cancer-associated immunogens, Thomsen-Friedenreich (T) and Tn, myelin basic protein, and organ-specific cancer neoantigens

Peripheral blood leukocytes from cancer patients exhibit nonadherence to glass as an index of antigen recognition when incubated individually with four distinct, soluble tumor-related substances. Crude cancer extracts, purified antigens, T and Tn, myelin basic protein (MBP), and organ-specific cancer neoantigens (OSN), all elicited narrow dose-dependent leukocyte adherence inhibition (LAI) response curves. The present study focused on the reasons for the narrow antigen dose-LAI response relationship. Between 9 and 20 pmol of antigens elicited the maximum number of nonadherent leukocytes; cleavage products of T antigen and the nonapeptide (T18) of MBP required about a 10-fold increase in molar concentration for the same LAI response. When crude cancer extracts were combined with pure antigen or the pure antigens were combined at concentrations shown to give maximum LAI responses, the positive LAI responses were negated. The chemoattractant LTB4 at 10(-11) M triggered maximum LAI. But when MBP was added with the LTB4 at progressively increasing concentrations, there was dose-dependent negation of LAI. The magnitude of LAI depended on the total amount of mediator released rather than the rate of release. When leukocytes from cancer patients were incubated with optimum to high concentrations of MBP, the supernatants contained a mediator that gave similar bell-shaped dose-LAI responses on control leukocytes indicating that leukocytes from cancer patients react to a much broader range of antigen concentration than indicated by the LAI assay alone. High antigen dose negated LAI because of excess mediator production. Antigen-generated mediators had a biphasic effect inducing nonadherence and then adherence of leukocytes.