Induction and reversal of immune paralysis in vitro.

Induction of the immune response can only be completed after antigen is removed from the cellular environment. Primed rabbit lymph node fragments were cultured in vitro with 5 mg/ml BSA. If antigen was removed from the fragments 2 hr later, they produced a normal anti-BSA response, which was first evident 5 days later. If antigen removal was delayed for 3 days, the onset of the response was postponed for 2 to 3 days. Pulses with BUDR marked the periods of cell proliferation in both sets of cultures, and established that the postponement of antibody production was preceded by a postponement in the wave of proliferation among precursors of antibody forming cells. The similarity in avidity of antibody-containing fluids from normal and postponed cultures support the idea that the same cell population produced the response in each case. It was concluded that a reversible state of paralysis could be instituted in antigen-responsive cells, and this state did not depend upon cell-killing. The widespread incidence of temporary paralysis as an early aspect of the immune response was discussed.

The X-Y-Z scheme of immunocyte maturation. IV. The exhaustion of memory cells.

A set of conditions has been described under which primed rabbit lymph nodes produce a secondary antibody response upon in vivo stimulation with a large dose of antigen, but are subsequently "exhausted;" that is, lymph node cultures prepared at intervals following the booster injection cannot be re-stimulated to display tertiary responses. Rabbits given 100-fold less antigen in the booster inoculum were able to give a tertiary response upon in vitro challenge. The system used permits neither induction nor continuation of a primary response to BSA in vitro. Since it could be demonstrated that no memory cells were generated by the booster injection within the intervals between in vivo injection and culture, the tertiary response in nonexhausted nodes must have been due to residual memory cells which remained untriggered by the in vivo booster injection. The unresponsive state was not caused by antibody feedback. These results are interpreted to mean that a population of memory cells can be exhausted by a supraoptimal dose of antigen, rendering the node temporarily incapable of further response. This implies that long-lived memory is not due to asymmetric division of memory cells. The source and fate of memory cells is discussed with regard to this evidence.

The X-Y-Z scheme of immunocyte maturation. V. Paralysis of memory cells.

A concentration of 5 mg/ml bovine serum albumin (BSA) prevents the in vitro elicitation of a secondary response in primed rabbit popliteal lymph nodes, if it is left in contact with the node fragments for the first 6 days of culture. No antibody formation can be detected at any time during the culture period in most cases, although occasional fragments are resistant to inhibition. Reducing the exposure time to the first 3 days of culture delays the peak of the antibody response. The inhibition is antigen specific. Reconstruction experiments demonstrate that the inhibition is not due to antigen masking of the antibody. Even shortly after optimal stimulation, the addition of 5 mg/ml BSA to the fragments was not able to prevent a normal antibody response. The implications of these findings are that (a) a high antigen concentration suspends the memory cell in a reversibly paralyzed state, (b) memory cells have a heterogeneous susceptibility to inhibition, (c) once induced, the antibody response cannot be inhibited by antigen overloading, (d) unresponsiveness in a primed animal can be due to either exhaustion of the memory cell population or paralysis of the memory cell.

In vitro studies of poison oak immunity. II. Effect of urushiol analogues on the human in vitro response.

Studies were performed to ascertain the effect of urushiol analogues on the in vitro lymphocyte blastogenesis elicited by urushiol in peripheral blood lymphocytes taken from individuals sensitized to poison oak or ivy. Urushiol is a mixture of alkylcatechols composed of a catechol ring coupled to mono-, di-, or tri-unsaturated C-15 or C-17 carbon side chains. Each of these two moieties, catechol ring and side chain, was tested for its role in eliciting reactivity. Analogues tested represented the catechol ring (3-methylcatechol), the mono- or di-unsaturated side chain (oleic or linoleic acid), and the saturated side chain coupled to a catechol ring (pentadecylcatechol), a blocked catechol ring (heptadecylveratrole), or a resorcinol (pentadecylresorcinol). Urushiol with a blocked catechol ring (urushiol dimethyl ether) was also included. Of these, only pentadecylcatechol evoked reactivity in sensitized lymphocytes, and this reactivity was only a fraction of that evoked by urushiol. This suggested that the system has some requirement for the side chain, and that the catechol ring is critical for reactivity. This was further investigated by testing the ability of some of these analogues to inhibit urushiol-specific blastogenesis. No inhibition was noted with compounds bearing the saturated side chain with modified ring structures (pentadecylresorcinol and heptadecylveratrole). However, both 3-methylcatechol and pentadecylcatechol (at equimolar concentrations) blocked reactivity. The results of our experiments suggested that although both the side chain and the catechol ring are required for reactivity, the latter is most critical. Unsaturation in the side chain is important for maximal reactivity because the saturated catechols were only partially as active as the urushiol oil. There may be a greater dose requirement for the catechol ring than for the side chain.

Tumor-specific cell-mediated immunity in household contacts of cancer patients..

Patients with osteogenic sarcoma (and related tumors), hypernephroma, and breast carcinoma, and their household contacts were tested for tumor-specific cell-mediated immunity against these tumors with the use of a short-term chromium-51 release assay. This assay, reproducible over many months and well-correlated with the clinical course of the patients, was used to demonstrate that household contacts of patients with osteogenic sarcoma and breast carcinoma have specific immunity against the tumor type with which they have been in contact. In both types of tumors, the range of cytotoxicity values produced by lymphocytes from the household contacts was significantly higher than that of the normal population. The incidence of immunity was much higher in household contacts of patients with breast carcinoma than in those of patients with osteogenic sarcoma. Immunity was found with equal frequency in men and women, as well as in genetically and nongenetically related household contacts (guardians, adopted children, spouses). Immunity against hypernephroma was not demonstrated in either patients with hypernephroma or their household contacts.

In vitro studies of poison oak immunity. I. In vitro reaction of human lymphocytes to urushiol.

Poison oak, ivy, and sumac dermatitis is a T-cell-mediated reaction against urushiol, the oil found in the leaf of the plants. This hapten is extremely lipophilic and concentrates in cell membranes. A blastogenesis assay employing peripheral blood lymphocytes obtained from humans sensitized to urushiol is described. The reactivity appears 1--3 wk after exposure and persists from 6 wk to 2 mon. The dose-response range is narrow, with inhibition occurring at higher antigen concentrations. Urushiol introduced into the in vitro culture on autologous lymphocytes, erythrocytes and heterologous erythrocytes produces equal results as measured by the optimal urushiol dose, the intensity of reaction, and the frequency of positive reactors. This suggests that the urushiol is passed from introducer to some other presenter cell. Although the blastogenically reactive cell is a T cell, there is also a requirement for an accessory cell, found in the non-T-cell population, for reactivity. Evidence is presented that this cell is a macrophage.

Osteogenic sarcoma. Immunologic parameters before and during immunotherapy with tumor-specific transfer factor.

18 patients with osteogenic sarcoma were followed by serial measurements in vitro of tumor-specific cell-mediated cytotoxicity and of "active" and total rosette-forming T-cells. 13 of these patients have had or are currently receiving injections of osteogenic sarcoma-specific dialyzable transfer factor derived from healthy donors. In three patients with very small lesions, cytotoxicity was high before amputation and decreased within 2 mo after removal of tumor. Cytotoxicity was low at time of diagnosis in all patients with large tumor masses. The cytotoxicity of the patients' lymphocytes increased after administration of tumor-specific transfer factor in all patients so treated. Patients receiving nonspecific transfer factor showed evidence of declining cell-mediated cytotoxicity. Tumor-specific transfer factor may produce an increase in cell-mediated cytotoxicity to the tumor in patients with osteogenic sarcoma. This possibility is suggested by the pain and edema that occurred in the area of the tumor in patients who had metastatic disease when therapy was started and by lymphocytic infiltrates in the tumor, as well as by the increase in cell-mediated cytotoxicity and the increase in percentage of active rosette-forming cells from subnormal to normal. Serial measurements of cell-mediated cytotoxicity are helpful in monitoring the efficacy of transfer factor and other modes of therapy in these patients, and these measurements are the best available criteria for selection of donors of tumor-specific transfer factor.

Antigenic differences among osteogenic sarcoma tumor cells taken from different locations in human tumors.

Sections were taken from the center, midzone, and margin of four human osteogenic sarcomas and one fibrosarcoma. Single-cell suspensions of tumors were examined in an indirect immunofluorescence assay with autologous or homologous anti-osteogenic sarcoma antisera as the intermediate reactant and fluorescein-labeled anti-human IgG as the final reactant. Cells were stained under conditions in which the fluorescence intensity was directly proportional to the density of the tumor-associated antigen on these cells. The density of tumor-associated antigen on cells from the center of the five tumor masses was low; cells from the midzone had intermediate levels of tumor antigen density, and cells at the margin had the highest levels. Similar preparations stained with polyspecific anti-HLA antisera did not demonstrate such a gradient. Since osteogenic sarcomas grow outward from the center, with the outer margin populated by the youngest cells, these results suggest that the oldest cells in the tumor bear the least tumor antigen, and the youngest tumor cells have the most. This is not compatible with theories which postulate that the immune system modulates the growth of a tumor so that only the least antigenic cells are allowed to grow. Alternative mechanisms are discussed.

Quantitative immunofluorescence studies of the tumor antigen-bearing cell in giant cell tumor of bone and osteogenic sarcoma.

Tumor-associated antigen was found by reacting sera from two patients with giant cell tumor of bone with cells derived from their tumors, using autologous serum as intermediate reactant and fluorescein-conjugated goat anti-human IgG as final reactant. Approximately 40% of the plump, spindle-shaped cells that formed the background stroma of these tumors possessed the antigen; however, it was not present on giant cells. Fluorescence was much greater than that on similarly stained cells from 4 osteogenic sarcomas, suggesting that the antigenic density on cells from giant cell tumor was greater than that on cells from osteogenic sarcoma. Antibodies in sera from giant cell tumor patients and osteogenic sarcoma patients showed specific cross-reactivity. Stromal cells of giant cell tumors were established in culture and retained tumor-associated antigen, whereas giant cells failed to divide and detached from the flask within two weeks. Intensity of fluorescence (antigenic density) decreased with progressive passage levels, but a larger percentage of cells showed fluorescence. At the tenth passage, all cells bore tumor-associated antigen. Cultured cells that were injected s.c. into mice formed progressively growing nodules, the cells of which were morphologically indistinguishable from stromal cells of the original tumor; all cells retained tumor-associated antigen, but antigenic density had decreased to about one-seventh of the value found originally. No giant cells were present in the nodules.

Sensitivity and selectivity of ricin toxin A chain-monoclonal antibody 791T/36 conjugates against human tumor cell lines.

Ricin toxin A chain (RTA) was conjugated to monoclonal antibody 791T/36, which was raised originally against human osteogenic sarcoma cell line 791T. The resultant conjugates were characterized and tested for cytotoxicity against a panel of human tumor cell lines representing a defined range of antigenicity with regard to 791T/36. Conjugates were highly cytotoxic for cells expressing high antigen density, inhibiting cell survival at RTA concentrations three to four orders of magnitude lower than that possible with RTA alone. Cytotoxicity of conjugates diminished with decreasing 791T/36 antigen concentration on target cells, but significant effects were seen against cells of low or intermediate antigenicity. Cytotoxicity could be blocked specifically by excess 791T/36 antibody, clearly indicating that antigen binding was a necessary part of the mechanism of action. Comparison with drug-antibody conjugates indicated that RTA immunotoxins are much more active, but discriminate less readily than drug-antibody conjugates between cells of different antigenicity. It is suggested that these properties be taken into account with regard to practical application and future development.

Inhibition of growth of human tumor xenografts in athymic mice treated with ricin toxin A chain-monoclonal antibody 791T/36 conjugates.

Immunotoxin constructed by conjugating ricin A chain to monoclonal antibody 791T/36 specifically inhibits growth of human tumor xenografts which express the gp72 antigen recognized by the antibody component. Dose schedule tests showed that the major response was obtained during the first 5 days of treatment and further prolonged treatment did not improve therapy. Expression of gp72 antigen on tumor cells derived from xenografts in immunotoxin-treated mice was not markedly altered indicating that treatment did not lead to the expansion of tumor antigen deficient tumor cells. The experiments indicate that treatment for short duration with immunotoxin may be the most effective protocol.

Phase I study of monoclonal antibody-ricin A chain immunotoxin XomaZyme-791 in patients with metastatic colon cancer.

Monoclonal antibody 791T/36, recognizing a Mr 72,000 antigen on the surface of colon carcinoma cells, has been used to construct an immunotoxin by conjugating to it the ribosomal inhibitor protein, ricin toxin A chain. The antibody 791T/36 has been shown to bind to membranes of freshly disaggregated tumor cells from human colon tumors, and to localize in tumors in vivo. Subacute toxicology testing in rats receiving immunotoxin i.v. showed, at highest doses, weight loss, decreased serum albumin, and hepatocyte vacuolization without elevation in liver function tests. A Phase I dose escalation study was carried out in which 17 patients with metastatic colorectal cancer were treated with doses of immunotoxin ranging from 0.02 to 0.2 mg/kg/day in 1-h i.v. infusions for a 5-day course. Side-effects included a composite of signs and symptoms thought to be generic to ricin A chain immunotoxins, including decreased serum albumin, mild fever, and flu-like symptoms, all being reversible. Two additional findings, reversible proteinuria and mental status changes, were also noted which may be characteristic of this immunotoxin. By 10-20 days after therapy, most patients developed IgM and IgG antibodies against both the ricin toxin A chain and the immunoglobulin portion of the immunotoxin, which were asymptomatic. A strong anticombining site antibody response was seen. Biological activity manifest as mixed tumor regression was seen in five patients.

Biodistribution of ricin toxin A chain-monoclonal antibody 791T/36 immunotoxin and influence of hepatic blocking agents.

Immunotoxin constructed by conjugating ricin A chain to monoclonal antibody 791T/36 has a markedly altered biodistribution when compared to unconjugated antibody. This is principally manifest as hepatic uptake of immunotoxin which appears to be controlled by the ricin A chain (RTA) moiety. This was established by comparing the blood survival and organ distribution of immunotoxin with that of ricin A chain and free antibody using preparations in which either the RTA or antibody, alone or as components of the immunotoxin, was radiolabeled. Gel filtration chromatography of sera from immunotoxin treated animals demonstrated a preferential blood clearance of immunotoxin with high RTA-antibody ratio. Hepatic uptake is dependent upon Kupffer cell recognition of mannose-containing oligosaccharide structures on the RTA moiety of immunotoxin. Mannose-containing blocking agents given with immunotoxin were shown to prolong circulation time of the immunotoxin in blood including those species with higher RTA-monoclonal antibody ratios and reduce liver uptake. Effective blocking agents include ovalbumin, ovomucoid, and mannosyl-lysine (Man3Ly2). These studies demonstrate that agents specifically inhibiting hepatic uptake of immunotoxin significantly alter biodistribution and may improve their therapeutic efficacy.

Endocytosis of immunotoxin-791T/36-RTA by tumor cells in relation to its cytotoxic action.

Ricin A chain immunotoxin constructed with monoclonal antibody 791T/36, which recognizes a tumor associated glycoprotein Mr 72,000 antigen present on sarcomas and colon and ovarian cancer cells, is cytotoxic for cell lines from tumors expressing this antigen. Incubation of sarcoma 791T cells with immunotoxin for only 5 min is sufficient to produce greater than 95% inhibition of tumor cell growth. Papain treatment of these cells to remove immunotoxin from the cell surface indicated that the cell surface acts as a reservoir for continued internalization of immunotoxin over several hours, but even so, 50% inhibition of cell survival was produced over a 2- to 3-h period. Analysis of the rate of endocytosis demonstrated that 30-50% of cell bound immunotoxin was internalized over a 180-min period. This was primarily dictated by the antibody moiety, regardless of the degree of conjugation to ricin A chain. This rate is much slower than that of other cell surface ligands such as transferrin. Cell cytosol acidification experiments were performed to determine whether this immunotoxin was internalized by clathrin coated pits, which is relatively rapid, or by smooth pits, which is slower, and the results indicated the latter mechanism is almost exclusively used. Intracellular trafficking of antibody 791T/36, conjugated to human serum albumin-tetramethylrhodamine was investigated by flow cytometry. The movement of the conjugate into the lysosomal compartment was delayed so that degradation products were only detected after a lag phase of 30-60 min. The lack of potentiator dependence of 791T/36 immunotoxin is in keeping with these findings.

A phase I/II study of trichosanthin treatment of HIV disease.

Trichosanthin, a ribosomal inhibitor protein, blocks HIV replication in lymphocytes and macrophages. This agent was used to treat 51 patients with advanced HIV disease in a dose-escalation study in which three injections were administered over a 9-21-day period in a dose range of 10-30 micrograms/kg per injection. The maximum tolerated dose was estimated to be 30 micrograms/kg. Reversible but severe fatigue and myalgias were the major dose-limiting side-effects; mild leucocytosis and elevations in serum transaminases were noted and were reversible. Non-dose-related reversible mental status changes were seen in six patients and were considered to be associated with the drug. This was usually manifest as dementia, but progressed to coma in two patients. This reversed, but the sequelae resulted in death in one patient. Decreases in serum p24 antigen levels were noted 1 month after the first infusion in 10 of 18 patients who entered the study with elevated levels; one converted to negative. Values usually remained low to the end of the study period (2 months). In those patients with CD4+ cell levels greater than 50 x 10(6) cells/l significant decreases in sedimentation rate and increases in CD4+ cell numbers were also noted. These changes were found at all dose levels but only in patients receiving three infusions.

Induction of tolerance to poison ivy urushiol in the guinea pig by epicutaneous application of the structural analog 5-methyl-3-n-pentadecylcatechol.

Previous studies have established that epicutaneous application of 5-methyl-3-n-pentadecylcatechol (5-Me-PDC), a synthetic analog of a poison ivy urushiol component, leads to immune tolerance to 3-n-pentadecylcatechol (PDC) in mice. The induction of tolerance by 5-Me-PDC may be mediated by a protein conjugate formed via selective reaction of thiol nucleophiles present on the carrier macromolecule with the corresponding o-quinone derived from the parent catechol. In order to examine further the tolerogenic properties of 5-Me-PDC, we have extended our studies to the guinea pig, the generally accepted experimental species for the study of contact allergy. The results have established that specific immune tolerance to poison ivy urushiol is induced following 2 epicutaneous applications of the PDC analog. Furthermore, we were able to show that the treated animals remained tolerant for at least 6 weeks, a period of time comparable to that observed following the intravenous administration of the O,O-bis-acetyl derivative of PDC. The data point to the possibility of developing a therapeutically effective topical tolerogen for poison ivy contact dermatitis.

Suppression of urushiol-induced delayed-type hypersensitivity responses in mice with serum IgG immunoglobulin from human hyposensitized donors.

Serum IgG immunoglobulin fractions from human subjects hyposensitized to poison ivy/oak by oral administration of urushiol suppressed the induction of delayed-type hypersensitivity (DTH) responses in mice to this hapten. This suppressive activity was hapten specific because it did not modify DTH responses to dinitrofluorobenzene (DNFB). Absorption of human serum with lymph node cells from urushiolsensitized but not DNFB-sensitized mice removed the suppressive activity, suggesting that anti-idiotypic antibodies reacting with T-cell receptors are involved.

Regulation of murine contact sensitivity to urushiol components by serum factors.

Mice epicutaneously painted with components of poison ivy urushiol oil exhibit contact sensitivity (as detected by ear swelling reactions) that persist for about 25 days. Sera taken from mice at times when the contact sensitization response is waning suppressed the induction of sensitization to 3-n-pentadecylcatechol (PDC), a urushiol component, in recipients. The suppressive serum factor was present in greatest amount 25 days after sensitization, but was no longer detectable 40 days post sensitization. Suppression was antigen-specific, absorbed out with PDC-immune, but not normal lymph node cells, and transferable with a single 0.6 ml dose 7 days prior to sensitization of recipients. Suppression was transferable by the purified IgG fraction of desensitized mice. Results indicate that contact sensitivity to urushiol in mice is regulated by serum factors.

Regiospecific attack of nitrogen and sulfur nucleophiles on quinones derived from poison oak/ivy catechols (urushiols) and analogues as models for urushiol-protein conjugate formation.

Attempts to characterize potential biologically important covalent interactions between electrophilic quinones derived from catechols present in poison oak/ivy (urushiol) and biomacromolecules have led to the analysis of model reactions involving sulfur and amino nucleophiles with 3-heptadecylbenzoquinone. Characterization of the reaction products indicates that this quinone undergoes regiospecific attack by (S)-N-acetylcysteine at C-6 and by 1-aminopentane at C-5. The red solid obtained with 1-aminopentane proved to be 3-heptadecyl-5-(pentylamino)-1,2-benzoquinone. Analogous aminobenzoquinones were obtained with the quinones derived from the 4- and 6-methyl analogues of 3-pentadecylcatechol. All three adducts absorbed visible light at different wavelengths. When the starting catechols were incubated with human serum albumin almost identical chromophores were formed. These results establish that cathechols responsible for the production of the poison oak/ivy contact dermatitis in humans undergo a sequence of reactions in the presence of human serum albumin that lead to covalent attachment of the catechols to the protein via carbon-nitrogen bonds. Estimations of the extent of this binding indicate that, at least with human serum albumin, the reaction is quantitative.

A phase II study of effect of addition of trichosanthin to zidovudine in patients with HIV disease and failing antiretroviral agents.

Patients infected with HIV, including those with AIDS-related complex and AIDS, and failing treatment with antiretroviral agents such as zidovudine, have been evaluated following addition of trichosanthin to the antiretroviral agent regimen. This ribosomal inhibitory protein is specifically cytotoxic for HIV-infected macrophages and lymphocytes. Ninety-three patients were treated with trichosanthin, using a schedule of weekly, then monthly, intravenous injections of 1.2 mg of drug in combination with antiretroviral agents, usually zidovudine. Side effects included myalgias, fevers, mild elevation in liver function tests, and mild-moderate anaphylactic reactions, which respond well to therapy with steroids and/or benedryl. Reversible mental status changes were noted in two patients, both receiving concomitant therapy with ddI. Clinical responses to trichosanthin treatment were monitored primarily by changes in laboratory parameters, particularly levels of CD4+ T lymphocytes. In the total population evaluated for efficacy (85 patients) there was a significant increase in CD4+ cell levels after initiation of trichosanthin therapy. A second analysis performed on 72 patients measured the rate of change of CD4+ cells during therapy, using an "area under the curve" analysis. During therapy there was a median increase of 1.2 cells/mm3/month. In patients in the top 25th percentile, this increase was greater than 8.4 cells/mm3/month. In 59 of the 72 patients, responses could also be monitored by comparing the rate of loss of CD4+ cell levels on antiretroviral agents (zidovudine or ddI) alone, during the year prior to initiation of trichosanthin, to the rate of change when trichosanthin was added to the treatment regimen. During the period before trichosanthin treatment (311 +/- 11.7 days) the median loss of CD4+ cells was 6.91 cells/mm3/month. Addition of trichosanthin to the treatment regimen resulted in a median gain of 1.1 CD4+ cells/mm3/month.