The comparison of methods for determination of humoral and cell-mediated immunity in patients with breast cancer.

We observed the immunological answer to antigens obtained from the human malignant breast tumor and from the blood of inbred mice strain C3H/H2K infected by LDH virus. We compared the modified ELISA method for humoral immunity with the leukocyte adherence inhibition (LAI) assay for cell-mediated immunity. The modified ELISA method is suitable for early diagnosing and monitoring antibodies in a malignant breast tumor simultaneously with senological examinations which include mammography and clinical examinations, because the antibodies are determined in a high number of samples by single application.

Hemodialysis leukopenia. Pulmonary vascular leukostasis resulting from complement activation by dialyzer cellophane membranes.

Acute leukopenia occurs in all patients during the first hour of hemodialysis with cellophanemembrane equipment. This transient cytopenia specifically involves granulocytes and monocytes, cells which share plasma membrane reactivity towards activated complement components. The present studies document that complement is activated during exposure of plasma to dialyzer cellophane, and that upon reinfusion of this plasma into the venous circulation, granulocyte and monocyte entrapment in the pulmonary vasculature is induced. During early dialysis, conversion of both C3 and factor B can be demonstrated in plasma as it leaves the dialyzer. Moreover, simple incubation of human plasma with dialyzer cellophane causes conversion of C3 and factor B, accompanied by depletion of total hemolytic complement and C3 but sparing of hemolytic C1. Reinfusion of autologous, cellophane-incubated plasma into rabbits produces selective granulocytopenia and monocytopenia identical to that seen in dialyzed patients. Lungs from such animals reveal striking pulmonary vessel engorgement with granulocytes. The activated complement component(s) responsible for leukostasis has an approximate molecular weight of 7,000-20,000 daltons. Since it is generated in C2-deficient plasma and is associated with factor B conversion, it is suggested that activation of complement by dialysis is predominantly through the altermative pathway.

Spectrum of immunodeficiencies with Hodgkin's disease.

The role of six suppressor mechanisms upon T and B cell responses was studied on 17 untreated patients with Hodgkin's disease. Proliferative hyporesponsiveness to mitogen was greatly impaired in 8 of the 13 patients. 10 of these patients had an excessive degree of suppression by cells that adhered to foreign surfaces. Suppression by adherent cells correlated with impairment of proliferative responses and, in some instances, suppression was largely inhibited with indomethacin. Likewise, adherent cells suppressed immunoglobulin synthesis. A correlation was evident between suppression of T and B cell responses by adherent mononuclear leukocytes from individual patients. This suppression coincided with elevated percentages of monocytes in the patient mononuclear cell preparations. This excess of monocytes was not the result of a circulating monocytosis. The monocyte excess may have been acquired during isopyknic cell separation. A second form of suppression was observed in 5 of the 11 patients affected by a lymphocyte that neither adhered to glass wool nor required preactivation. It did not inhibit allogeneic lymphocytes, which contrasts with the suppressor abnormality of monocytoid cells.

Cultured human endothelial cells stimulated with cytokines or endotoxin produce an inhibitor of leukocyte adhesion.

Activation of cultured human endothelial cells (HEC) by inflammatory stimuli, such as interleukin 1 (IL-1), tumor necrosis factor (TNF), and bacterial endotoxin (lipopolysaccharide, LPS), increases their surface adhesiveness for blood leukocytes and related cell lines. We now report that activated HEC also generate a soluble leukocyte adhesion inhibitor (LAI), which accumulates in conditioned media from IL-1-, TNF-, or LPS-treated, but not sham-treated, HEC cultures. LAI significantly inhibits the adhesion of PMN and monocytes to activated, but not unactivated, HEC. In contrast, LAI has no effect on the adhesion of lymphocytes, the promyelocytic cell line HL-60 or the monocyte-like cell line U937 to HEC monolayers. LAI appears to act directly on the leukocyte, but does not inhibit either agonist-induced responses in PMN (membrane depolarization, changes in cytosolic calcium concentration, superoxide production) or PMN attachment to serum-coated plastic surfaces. Endothelial generation of LAI is blocked by actinomycin D but not by aspirin or indomethacin. Preliminary biochemical characterization indicates that LAI is a soluble, protein-containing molecule that is heat- and acid-stable. Fractionation by HPLC gel filtration yields a single peak of LAI activity (14,000 less than Mr greater than 24,000). Thus, in addition to proadhesive cell surface changes, the endothelium may also actively contribute to the regulation of endothelial-leukocyte interactions at sites of inflammation in vivo through the production of soluble adhesion inhibitors such as LAI.

The direct thrombin inhibitor melagatran counteracts endotoxin-induced endothelial leukocyte adherence and microvascular leakage in the rat mesentery. Rationale for the treatment of inflammatory disorders beyond sepsis?

The activation of the coagulation system in the course of an inflammatory reaction impairs the function of the microcirculation. By means of intravital videomicroscopy the effect of the direct thrombin inhibitor melagatran on endotoxin-induced microvascular permeability and leukocyte adhesion to microvascular endothelium of rat mesentery was evaluated. Secondly, plasma concentrations of melagatran or interleukin-6 in response to endotoxin or after treatment with melagatran respectively, were determined. Male Sprague-Dawley rats (300-400 g bw) were infused with 0.5 mg/kg lipopolysaccharide (LPS) (E. coli O55:B5) over 80 minutes. Vascular leakage was detected with FITC-marked rat serum albumin by fluorescence microscopy and evaluated by grey value analysis with a computer assisted image processing system. Light microscopy was used to evaluate the adherence of leukocytes to the vessel wall. Two treated groups received either 0.3 or 0.6 mg/kg bw melagatran iv in addition to LPS-infusion. The observation period was 3 hours after the beginning of LPS infusion. Groups of animals not infused with LPS or solely treated with melagatran (0.3 or 0.6 mg/kg) served as controls. Infusion of LPS led to a significant increase of microvascular permeability, leukocyte adherence and thrombin-antithrombin complex plasma concentration compared to unstimulated controls. These effects were significantly reduced by melagatran at both dosage levels. Elevated plasma concentrations of melagatran were observed in animals infused with endotoxin and higher plasma levels of interleukin-6 were found in endotoxemic animals treated with melagatran. The results indicate that thrombin is one of the most important clotting enzymes involved in inflammatory microvascular disturbance. Moreover, it should be clarified whether direct thrombin inhibitors themselves play a role within the immune response to endotoxin.

Regulation of cell-mediated immunologic reactivity to Moloney murine sarcoma virus-induced tumors. I. Cell and serum activity detected by leukocyte adherence inhibition.

Cell-mediated immunity and serum regulatory factors were studied in an in vitro system involving a spontaneously regressing, virus-induced tumor. Inbred BALB/c and CBA mice were inoculated with Moloney murine sarcoma virus and their peritoneal cells were tested for reactivity in leukocyte-adherence inhibition tests with extracts of syngeneic tumors. Sera from inoculated mice were tested for their effect on this reactivity. At the optimal dilution, tumor extracts induced significant reactions with cells from tumor-bearing mice (progressors) and from mice with regressed tumors (regressors); cells from normal mice and from mice with transplanted, chemically induced tumors were unaffected. Sera from progressor mice specifically blocked the reactivity of syngeneic cells. At the time of maximal tumor development, this blocking activity disappeared and the serum became unblocking: i.e., the regressor serum neutralized the blocking factor in progressor serum. The blocking or unblocking factors were tumor-specific; no cross-reactivity occurred with similar factors related to the chemically induced tumor. Normal cells were not significantly affected by exposure to blocking and unblocking sera. The development of cellular immune reactivity and serum factors detected in vitro corresponded to the cycle of tumor progression and regression observed in vivo.

Immune response to melanoma extracts in three melanoma-prone families.

Immune reactivity to melanoma extracts was measured by the leukocyte adherence inhibition (LAI) test in 40 members of 3 melanoma-prone families. The melanoma patients had a wide range of responsiveness to the extract, the highest responder being a 10-year survivor. As a group, family members (including spouses) without disease had significantly elevated LAI responses compared to those of unrelated controls (P less than 0.01). Within the families, members with close exposure to melanoma patients for 10 years or more had a significantly higher response to melanoma antigen than did members with 0-5 years of close exposure (P less than 0.05). Responses of spouses and members at high risk of developing melanoma (B-K mole syndrome) also correlated with length of exposure to patients, which suggests that the elevated LAI response was not genetically determined. The high frequency of positive responses to melanoma antigens in these families, particularly in spouses, suggests the presence of transmissible melanoma-associated material capable of immunizing persons in contact with melanoma patients.

Tumor-associated immunity in bovine ocular squamous cell carcinoma detected by leukocyte adherence inhibition microassay.

A microplate modification of the leukocyte adherence inhibition (LAI) assay was used with blood leukocytes from cattle with ocular squamous cell carcinomas (OSCC); control groups were cattle with ocular or cutaneous lesions (not carcinoma) and healthy normal cattle. For the assay, saline extracts of OSCC and skin from the same donor, lymphosarcoma, and mastocytoma (M) were used as antigens. Specific LAI reactivity to OSCC extract (but not to skin extract) was detected in 14 of 18 animals with squamous cell carcinoma. One animal with OSCC showed LAI reactivity to OSCC extract, but 1 clinically normal cow had LAI with the M-extract. Leukocyte adherence stimulation reactions with the various antigens were seen in all groups of animals.

TG cell involvement in the leukocyte adherence inhibition phenomenon.

Peripheral blood mononuclear cells (PBMC), T-cells, non-T-cells, TG cells, and TM cells were tested for leukocyte adherence inhibition (LAI) reactivity to purified protein derivative (PPD) in normal human volunteers to determine the reactive cell subfractions. T-cells were separated by sheep red blood cell rosetting and TM and TG cells by rosetting with anti-ox red blood cell (ORBC) IgM- and IgG-treated ORBC. Fresh PBMC, T-cells, and TG cells gave positive LAI reactivity to PPD in PPD-positive donors only. PBMC that had undergone 24-hour incubation or extensive washing, non-T-cells, and TM cells were unreactive. All cell subfractions were negative in PPD-negative donors. Indirect LAI assay was performed on the spent media from overnight incubation of unstimulated PBMC and also following a 1-hour incubation of the various cell subfractions obtained from a PPD-reactive donor with PPD. Positive reactions, indicating the release of an LAI factor (LAIF), were obtained with the spent media and with the T-cell and TG cell subfractions following PPD stimulation, whereas non-T-cell and TM cell subfractions yielded no reactivity. Incubation of sensitized T-cells with PPD in the presence of cycloheximide and preincubation of T-cells for 24 hours with cycloheximide before PPD exposure failed to inhibit LAI reactivity. These data suggest that T-cells, specifically TG cells, can release LAIF upon specific antigen stimulation with PPD, whereas non-T-cell and TM cell subpopulations fail to respond to the same stimulus. LAIF may also be released into the culture media with prolonged (overnight) incubation in artificial media or with extensive washing. Inhibition of protein synthesis during antigen exposure or up to 24 hours before antigen exposure does not appear to inhibit LAI reactivity.

Leukocyte adherence inhibition: tumor specificity of cellular and serum-blocking reactions in human melanoma, breast cancer, and colorectal cancer.

Blood samples were obtained from hosptial patients suspected of having cancer (colorectal carcinoma, breast carcinoma, or melanoma) or from patients after surgical treatment for these cancers. Leukocytes were tested for reactivity with appropriate tumor extracts by leukocyte adherence inhibition (LAI), without knowledge of the diagnosis. Leukocytes from each patient were tested with the specific related extract corresponding to the suspected tumor type and with at least one unrelated extract. Each patient's serum was tested for its effect on the adherence of autologous leukocytes with specific tumor extract. Detailed leukocyte adherence data are presented for each of the 110 patients. Of the 75 patients eventually diagnosed as having cancer of one of the above types, 67 (89.3%) gave positive specific LAI reactions and only 5 (6.7%) reacted nonspecifically. Of the 35 patients with benign, unrelated, or unidentified disease, 10 (28.6%) gave positive reactions; most of these were patients with benign breast disease reacting with breast tumor extract. Leukocyte reactions with related extracts were almost always blocked by autologous serum (only two exceptions). When tested with allogeneic leukocytes reacting with their corresponding extracts, sera never blocked leukocytes of a different tumor type; in some cases they also failed to block leukocytes of the same tumor type although autologous leukocytes were blocked.

Humoral antitumor immune responses in patients with breast cancer measured with the leukocyte adherence inhibition technique.

A modification of the hemacytometer leukocyte adherence inhibition (LAI) test was described. In this modification, 0.25% serum from patients with breast cancer was added with the relevant antigen to the assay system with the use of trypsinized leukocytes from control persons as indicator cells. The modified assay measured a humoral immune response. In studies of patients with untreated breast cancer (stages I and II) with the use of a KCl extract from a breast carcinoma or from MCF-7 cells as antigens, the modified LAI test was found to be at least as sensitive as was the ordinary test. In a blind study on sera collected from patients with breast cancer 0.5-2 years before the LAI measurements and stored at -20 degrees C, 15 of 18 (83%) patients had a positive response. Whereas the ordinary LAI test is limited to the use of fresh blood, the present test can be performed with small amounts of serum that can be frozen and stored.

Inhibition of leukocyte adherence by 3-M potassium chloride extracts of human colorectal tumors.

Leukocyte adherence inhibition (LAI) assays were performed to test whether peripheral blood leukocytes (PBL) from patients with colorectal cancer could be inhibited from attachment to a glass surface when tumor-associated antigens (TAA) of human colorectal tumors were present. The assays were performed with PBL from 41 patients with adenocarcinoma of the colon or rectum with 3-M KCl extracts of colorectal tumors. The results demonstrated the presence of colorectal TAA in the 3-M KCl extract of colorectal tumor materials. The reactivity of leukocytes from patients with a favorable prognosis showed an increasing LAI trend; the reactivity of leukocytes from patients with an unfavorable prognosis had a decreasing LAI trend. In individual patients, alterations in the sequential LAI results paralleled changes in the clinical status, which thus strongly indicated that the LAI assay could be of value in the assessment of antitumor immunity.

Regulation of cell-mediated immunologic reactivity to Moloney murine sarcoma virus-induced tumors. II. Nature of blocking and unblocking factors in serum.

Serum blocking factors, detected in sera of inbred CBA and BALB/c mice with progressing virus-induced tumors by their interference with leukocyte-adherence inhibition (LAI) with the use of syngeneic leukocytes, were found to be strain related: No blocking occurred with allogeneic leukocytes. Furthermore, the unblocking factors of regressor serum were also strain related: No unblocking of allogeneic progressor serum was observed. There was no requirement for H-2 compatibility between leukocytes and tumor cells from which the LAI-inducing antigen was obtained. Absorption of CBA blocking serum with anti-I-Jk antiserum (and with anti-Ik containing this antibody specificity) removed blocking activity; absorption of regressor serum did not reduce its unblocking activity. These observations indicated that certain blocking factors resembled other suppressor factors in possessing antigenic determinants coded by the I-J subregion of the major histocompatibility complex. Their strain restriction and antigen specificity suggested combination with reactive lymphocytes in blocking LAI. The properties of unblocking factors suggested they combine with blocking factors and resemble antiidiotypic antibodies.

Leukocyte adherence inhibition to myelin basic protein by cancer patients' T-lymphocytes in association with class II major histocompatibility antigens on monocytes.

Patients' leukocytes were shown to react consistently in tube leukocyte adherence inhibition (LAI) assays with myelin basic protein (MBP) at optimal concentration, whereas control leukocytes were nonreactive. Mononuclear cells from patients with cancer gave positive LAI reactions with MBP, but separated T-lymphocytes, monocytes, and neutrophils did not. The mononuclear cell LAI responses were blocked by monoclonal antibody (MAb) to monomorphic determinant of class II major histocompatibility complex (MHC) antigens and to T4+ (Leu-3a+) and T3+ (Leu-4+) T-cell differentiation antigens but not by antibody to class I MHC antigens or T8+ (Leu-2a+) antigens. MBP was thus recognized by helper T-cells, requiring presentation in association with class II MHC determinants on monocytes. MAb to class I and class II MHC antigens and to T8+ (Leu-2a+), T4+ (Leu-3a+), and T3+ (Leu-4+) differentiation antigens did not negate LAI mediated by peripheral blood lymphocytes to organ-specific cancer neoantigens (OSN) of crude extracts of allogeneic cancer, which had previously been shown to react with cytophilic antibody on allogeneic monocytes. When membrane OSN and leukocytes were autologous, T8+ (Leu-2a+) phenotypic T-cells also mediated LAI that was blocked by anti-T8 (Leu-2a) and anti-T3 (Leu-4). LAI induced by MBP was also negated by drugs that antagonize thromboxane-leukotriene biosynthesis, indicating that, in common with other LAI reactions, the terminal mediators of nonadherence are oxidative metabolites of arachidonic acid. In addition to clarifying the role of MBP in the cellular in vitro immunoreactivity of cancer patients, the present observations have important implications for theories of LAI. Sensitized leukocytes have different mechanisms for the recognition of antigens in different forms, and the antigen-stimulated leukocytes produce mediators that in a final common pathway induce nonadherence of surrounding cells through leukotriene-like metabolites.

T8 and T3 surface glycoproteins in human T-cell mediation of leukocyte adherence inhibition to extracts of autologous cancer.

Monoclonal antibodies (MAb's) [anti-Leu-1, anti-Leu-2a (T8), anti-Leu-3a (T4), and anti-Leu-4 (T3)] were used to elucidate the type of T-cell mediating leukocyte adherence inhibition (LAI) and the role of T-cell surface glycoproteins in LAI. T8+ (Leu-2a+) and T4+ (Leu-3a+) subtypes were isolated by panning. T8+ (Leu-2a+) cells showed LAI to extracts of autologous cancer, whereas the T4+ (Leu-3a+) subset had no LAI response. Moreover, MAb to the T8+ (Leu-2a+) glycoprotein negated T-cell LAI, but MAb to Leu-1+ or to T4+ (Leu-3a+) did not negate T-cell LAI to autologous cancer extracts. The T3+ (Leu-4+) differentiation also was essential for T-cell LAI to autologous cancer extracts since anti-Leu-4 (T3) negated the response. Since LAI to autologous cancer extracts depends ultimately on leukotriene and other oxidative metabolites of arachidonic acid generated by the T-cell binding tumor antigen, the effect of MAb on LAI induced by leukotriene B4 isomer III. 5(S), 12(R)-dihydroxy-6, 14-cis-8, 10-trans-eicosatetraenoic acid (LTB4) was examined. Authentic LTB4 induced nonadherence of 34% of adherent T-cells, and this effect was not negated by anti-Leu-1, anti-T8 (Leu-2a), or anti-T4 (Leu-3a). However, anti-T3 (Leu-4) abrogated LTB4-induced LAI of pure T-cells without any effect on the basic adherence properties of T-cells. The present findings indicated that LAI to autologous cancer extracts was mediated by T-cells of the T8+ phenotype when they recognize tumor antigen and polymorphic major histocompatibility complex determinants on autologous cancer membranes. Moreover, differentiation glycoproteins T8+ (Leu-2a+) and T3+ (Leu-4+) on the surface of the responding effector T-cells performed distinct biologic functions that enabled the tumor antigen to trigger T-cell LAI.

Postsurgical follow-up of colorectal cancer patients monitored by the leukocyte migration test.

Blood samples from more than 1,000 patients with colorectal cancer were tested in the leukocyte migration test (LMT) and by a radioimmunoassay for carcinoembryonic antigen (CEA) for evaluation of: 1) the correlation of LMT response to the status of disease and 2) the use of LMT and CEA determination in the monitoring of patients after surgical removal of the primary tumor. A total of 320 patients, which were tested repeatedly, constituted the key group of the investigation: Of these, 56 patients developed recurrences or metastases whereas 217 patients showed no sign of reactivated disease. The group of patients with persistent absence of disease showed test profiles of either persistent negativity (50%) or conversion of one or both parameters; persistently positive profiles were rarely encountered. Patients with recurrences and metastases showed either positive profiles or conversion of positivity; only 3 patients were negative in both tests. However, despite significant disease-related response of the LMT, the test is not recommended as an adjunct to postsurgical monitoring, because of its technical complexity.

Immunity to tumor-associated antigens in vasectomized men.

Leukocytes from 49 vasectomized and 57 age-matched nonvasectomized men were tested in the leukocyte adherence inhibition (LAI) assay for reactivity to sperm and various 3-M KCl human tumor extracts. Forty-four percent of the vasectomized men and 15% of the control group were reactive to the sperm antigen preparation (P less than or equal to 0.03). Similarly, a significantly higher percentage of vasectomized men responded to 3 of 5 tumor extracts tested: melanoma I (34.7 vs. 15.8%, P less than or equal to 0.04), squamous cell carcinoma (48.8 vs. 26.0%, P less than or equal to 0.04), and breast carcinoma (19.5 vs. 4%, P less than or equal to 0.04). Thirty percent of vasectomized versus 4% of the control group responded to more than two tumor antigens (P less than or equal to 0.03). The degree of LAI reactivity to each tumor extract was highly correlated with degree of antisperm LAI reactivity, and the degree of LAI responsiveness to one of the melanoma extracts was significantly correlated with antisperm antibody titer as measured by the sperm-immobilization assay. Furthermore, nonresponsive leukocytes from the control population converted to tumor antigen-responsive when incubated with sera from vasectomized LaI-positive men. Data from this study indicated that a large percentage of vasectomized men with sperm immunity were responsive to tumor-associated antigens in the LAI test and that antisperm antibodies or other serum factors played a role in this response. These results and the evidence that most tumor antigen-responsive cancer patients also have serum antibodies that reacted with sperm suggested that vasectomized men and cancer patients frequently responded to immunologically cross-reactive antigenic determinants present on both sperm and tumor cells.

Regulation of cell-mediated immunologic reactivity to Moloney Murine sarcoma virus-induced tumors. III. Further characterization of tumor-specific serum blocking factors.

Blocking factors in the sera of inbred CBA mice bearing either Moloney murine sarcoma virus-induced tumors or transplanted 3-methylcholanthrene-induced tumors specifically blocked in the leukocyte adherence inhibition reaction between tumor-sensitized peritoneal cells and tumor antigens. These factors were absorbed from serum by treatment with anti-Ia antibodies and could be eluted in a partially purified form. Chromatography on Sephacryl S-200 columns demonstrated blocking activity in fractions of high and low molecular weight (greater than 100,000 and 40,000--50,000, respectively) derived from whole sera or eluates. Active fractions from serum appeared to retain the tumor specificity of the original material.

Effect of a mouse mammary tumor virus-derived protein vaccine on primary tumor development in mice.

The vaccines used in this study were derived from purified murine mammary tumor virus (MuMTV) preparations. Approximately 60% of the protein fractions consisted of the major viral membrane glycoprotein gp52. Inoculation sc of 10 microgram MuMTV-S-derived vaccine significantly delayed the appearance of primary mammary tumors in GR and BALB/cfC3H mice (strains with high incidences of mammary cancer); in BALB/c and C3Hf mice, which have a moderate tumor incidence at an advanced age, this treatment resulted in a slight and substantial acceleration, respectively, of primary tumor development. The induced cellular immune reactivity for vaccination, as measured with the in vivo Winn test and the in vitro leukocyte adherence inhibition assay, was strongest in the GR strain as compared to the BALB/c strain. The titer of antibodies to tumor cells, as estimated by membrane immunofluorescence, was also higher in the GR strain. In BALB/cfC3H mice, the influence of different vaccination schemes with an MuMTV-O-derived protein vaccine on primary tumor development was studied. Before sc injection, the vaccine was precipitated on alum. A dose of 10 microgram vaccine resulted in a 61% decrease in tumor incidence. Two or five additional booster injections with 1 microgram protein vaccine had no beneficial effect, although the amount of antibody measured was increased after boosting.

Recognition of a purified Mr-40,000 organ-specific immunogen of human lung cancer by class II-restricted T-cells.

An Mr-40,000 polypeptide (p40) was purified from a lung cancer cell line on the basis of its antigenicity with leukocytes from lung cancer patients in an in vitro immunological assay of leukocyte adherence inhibition. Here, the cells and mechanism responsible for recognizing the purified p40 organ-specific cancer neoantigen (OSN) were studied. Buffy coat leukocytes from lung cancer patients showed a bell-shaped dose response with a peak response at 0.75 micrograms/assay. Mononuclear cells showed a similar response pattern, whereas pure T-cells were unreactive. Monoclonal antibodies (MAbs) to T-cell differentiation antigens T3 and T4 inhibited the response in a dose-dependent fashion, whereas anti-T8 had no effect, indicating T helper cells and their T3-antigen receptor complex recognized the antigen. MAbs to class II major histocompatibility complex (MHC) antigens also inhibited the response, whereas MAbs to class I MHC had no effect, indicating an important role for class II MHC antigens of monocytes. None of the MAbs inhibited the response to OSN on membrane fragments, which is mediated by antibody-dependent monocytes. Trypsin- or cyanogen bromide-cleaved p40 OSN triggered a response at the same concentrations as the intact molecule. The p40 OSN incubated with live leukocytes showed less than 30% proteolytic digestion. The results indicate that class II-restricted T-cells recognize, via their antigen-specific cell surface receptors, contiguous sequences within the immunogenic tumor molecule in the context of the molecule or peptides binding to class II transplantation antigens.