Cross-linking of major histocompatibility complex class II molecules by staphylococcal enterotoxin A superantigen is a requirement for inflammatory cytokine gene expression.

Staphylococcal enterotoxin A (SEA) has two distinct binding sites for major histocompatibility complex (MHC) class II molecules. The aspartic acid located at position 227 (D227) in the COOH terminus of SEA is one of the three residues involved in its interaction with the DR beta chain, whereas the phenylalanine 47 (F47) of the NH2 terminus is critical for its binding to the DR alpha chain. Upon interaction with MHC class II molecules, SEA triggers several cellular events leading to cytokine gene expression. In the present study, we have demonstrated that, contrary to wild-type SEA, stimulation of the THP1 monocytic cell line with SEA mutated at position 47 (SEAF47A) or at position 227 (SEAD227A) failed to induce interleukin 1 beta and tumor necrosis factor-alpha messenger RNA expression. Pretreatment of the cells with a 10-fold excess of either SEAF47A or SEAD227A prevented the increase in cytokine messenger RNA induced by wild-type SEA. However, cross-linking of SEAF47A or SEAD227A bound to MHC class II molecules with F(ab')2 anti-SEA mAb leads to cytokine gene expression, whereas cross-linking with F(ab) fragments had no effect. Taken together, these results indicate that cross-linking of two MHC class II molecules by one single SEA molecule is a requirement for cytokine gene expression.

Costimulation by B7-1 and LFA-3 targets distinct nuclear factors that bind to the interleukin-2 promoter: B7-1 negatively regulates LFA-3-induced NF-AT DNA binding.

We have characterized the regulation of nuclear factors involved in transcriptional control of the interleukin-2 (IL-2) promoter-enhancer activity in Jurkat T cells stimulated with superantigen presented on HLA-DR transfectants combined with the ligands LFA-3 (CD58) and B7-1 (CD80). Gel shift analyses showed that NF-AT was strongly induced in LFA-3-costimulated Jurkat T cells, suggesting that NF-AT is a key target nuclear factor for the CD2-LFA-3 pathway. Studies using HLA-DR-B7-1-LFA-3 triple transfectants showed that the LFA-3-induced NF-AT DNA binding activity was negatively regulated by B7-1 costimulation. In contrast, induction of a CD28 response complex containing only c-Rel proteins was seen after B7-1 costimulation. Both LFA-3 costimulation and B7-1 costimulation induced the AP-1 and NF-kappaB nuclear factors. Distinct compositions of the NF-AT complexes were seen in B7-1- and LFA-3-costimulated cells. LFA-3 induced primarily Jun-D, Fra-1, and Fra-2, while B7-1 induced June-D-Fos complexes. In contrast, AP-1 and NF-kappaB complexes induced in B7-1- and LFA-3-costimulated T cells showed similar contents. Transient transfection of Jurkat T cells with a construct encoding the IL-2 enhancer-promoter region (position -500 to +60) linked to a luciferase reporter gene revealed that B7-1 costimulation was required to induce strong transcriptional activity. Combined B7-1-LFA-3 costimulation resulted in a synergistic increase in IL-2 transcriptional activity. Multimers of the AP-1, NF-AT, NF-kappaB, and CD28 response elements showed distinct kinetics and activity after LFA-3 and B7-1 costimulation and revealed that B7-1 and LFA-3 converge to superinduce transcriptional activity of the AP-1, NF-AT, and CD28 response elements. Transcriptional studies with an IL-2 enhancer-promoter carrying a mutation in the CD28 response element site revealed that the activity was reduced by 80% after B7-1 and B7-1-LFA-3 costimulation whereas the transcriptional activity induced by LFA-3 was unaffected. Our data strongly suggest a selectivity in induction of nuclear factors by the CD2-LFA-3 and CD28-B7-1 pathways. This selectivity may contribute to regulation of the levels of IL-2 induced by LFA-3 and B7-1 costimulation and favor autocrine and paracrine T-cell responses, respectively.

3-methylcholanthrene: transient inhibition of the lytic step of mouse natural killer cells.

Female mice belonging to NMRI stock were inoculated neonatally with daily doses of 5 micrograms diethylstilbestrol (DES) or olive oil for the first 5 days after birth and in adult life with 20 or 100 micrograms 3-methylcholanthrene (MCA) or the vehicle tricaprylin only. The cytotoxic activity of spleen natural killer (NK) cells against YAC-1 target cells was studied in a 51Cr release assay at 2, 5, 10, or 15 days after the MCA injection. A dose of 20 micrograms MCA to neonatally olive oil-injected females did not influence the NK activity, whereas an injection of 100 micrograms MCA significantly depressed the NK activity to about half the value seen in controls. This suppression was transient, and the normal level was reached again 15 days after the injection. The depressed NK activity could not be related to humoral or cellular suppressor mechanisms. A study at the single-cell level revealed that the inhibition was due to interference with the lytic step of the NK cell without affecting target-binding capacity. In vitro exposure of spleen cells from MCA-treated animals to interferon fully restored the NK activity. Neonatal DES treatment resulted in a depressed NK activity in adult females to a level about half of that seen in olive oil-injected controls. The NK activity in DES-treated females was not influenced by either 20 or 100 micrograms MCA. The MCA-induced suppression of NK activity was discussed in relation to the earlier reported difference in incidence of MCA-induced sarcomas between DES-treated and control females after they were given 20 micrograms MCA in adult life, as well as in relation to the same incidence in the 2 groups treated with 100 micrograms MCA. The results are compatible with a significant role for NK cells during MCA carcinogenesis and indicate that a possible part of the tumorigenic effect of MCA is its early suppressive effect on NK cell activity.

Long-term effects of neonatal estrogen treatment on mitogen responsiveness of mouse spleen lymphocytes.

Neonatal female NMRI mice were given injections of olive oil (controls) or daily doses of corticosterone (10 micrograms), 17 beta-estradiol (10 micrograms), or diethylstilbestrol (DES) (0.01, 0.1, 1, or 5 micrograms) for the first 5 days after birth. The 5-micrograms dose of DES resulted in a persistently reduced in vitro mitogen response to concanavalin A or bacterial lipopolysaccharide of spleen lymphocytes from 6-, 10-, and 18-week-old or 17-month-old females. DES injections from day 6 through day 10 did not influence the later mitogen response. Treatment of ovariectomized 10-week-old females with 5 micrograms DES for 5 days resulted in a tendency to a reduced mitogen response (not statistically significant) 24 hours after the last DES injection. Four weeks later, the mitogen response was the same in experimental and control females. Different possible mechanisms for the persistent effect on the mitogen response are discussed. Neonatal DES treatment not only resulted in persistent changes in the cervicovaginal epithelium and in the hypothalamic-pituitary gland control system but also in the spleen lymphocyte mitogen response. The altered mitogen response should be a stimulus for a detailed analysis of the immune system in women exposed to DES during fetal life, some of whom develop later in life clear cell adenocarcinoma of the uterine cervix and vagina.

Effects of the immunomodulator LS 2616 on growth and metastasis of the murine B16-F10 melanoma.

The carboxamide-quinoline LS 2616 is a novel immunomodulator augmenting natural killer (NK) cell activity and T-lymphocyte related effector functions. To investigate the possible usefulness of LS 2616 in immunotherapy of tumors, the effect of the substance on growth and metastasis of the B16-F10 melanoma in syngeneic C57BL/6 mice was investigated. Treatment with LS 2616 from the time of s.c. inoculation of B16-F10 cells significantly reduced tumor take. Continuous treatment of mice with LS 2616 initiated 4 days prior to i.v. injection of tumor cells reduced the number of pulmonary metastases by 85%. When treatment with LS 2616 was started 4 days after i.v. injection of tumor cells, a time when established tumor foci were readily detectable in the lungs, a significant reduction in the number of pulmonary metastases resulted. LS 2616 significantly reduced the number of spontaneous pulmonary metastases developing from a B16-F10 tumor growing in the footpad. When treatment with LS 2616 was initiated after the establishment of grossly visible spontaneous pulmonary metastases, no significant effect on the number of metastases was found after 2 weeks of treatment. However, combined treatment with a dose of cyclophosphamide which in itself was ineffective resulted in a statistically significant 70% reduction in the number of remaining pulmonary metastases. Injection of antibodies to asialomonoganglioside which strongly reduce NK cell activity in various organs was used as a probe for the involvement of NK cells in the effects of LS 2616 on the B16-F10 tumor. The therapeutic efficiency of LS 2616 on tumor take when given from the time of s.c. inoculation, on the number of i.v. induced pulmonary metastases when treatment was started before tumor cell injection, as well as the spontaneous development of pulmonary metastases during exposure to the substance was abrogated by simultaneous injection with antibodies to asialomonoganglioside. In contrast, the beneficial effects of LS 2616 on already established i.v. produced or spontaneous pulmonary metastases were unaltered in mice made NK cell deficient by injection of anti-asialomonoganglioside antibodies. In conclusion, LS 2616 has potent antitumor activities mediated by NK cells as well as non-NK cell related defense mechanisms.

Effects of in vitro hyperthermia on human natural killer cells.

The present study demonstrates that human natural killer (NK) cells isolated from peripheral blood of normal individuals are highly sensitive to hyperthermia. The effect was time and dose dependent, and treatment of peripheral blood lymphocytes at 42 degrees for 1 hr almost completely abolished NK activity. The effect was not a consequence of cell death since only a small decrease in cell viability was observed and the viability of density gradient fractions enriched for NK activity was normal. Analysis of NK activity at the single-cell level by application of a conjugation assay in agarose revealed that hyperthermia interfered with target cell binding as well as the lytic cycle. Attempts to rescue NK activity after hyperthermia treatment by incubation overnight with human alpha-interferon or activation in mixed leukocyte culture was unsuccessful, indicating that even pre-NK cells are heat sensitive. In contrast, the proliferative response to alloantigens in mixed leukocyte culture and to the T-cell mitogen concanavalin A was unaffected. Hyperthermia exposure of cytotoxic T-lymphocyte generated in mixed leukocyte culture immediately before assay against allogeneic blast cells strongly inhibited their activity. Some alterations in the kinetics of stimulation with the B-cell mitogen Staphylococcus aureus bacteria were observed after heat exposure although maximal stimulation was at control levels. Thus, NK cells, including their precursors, seem to be preferentially sensitive to hyperthermia among various lymphoid subclasses.

Neonatal estrogen treatment and epithelial abnormalities in the cervicovaginal epithelium of adult mice.

Female NMRI mice were given injections of different doses of 17 beta-estradiol, 17 alpha-estradiol, diethylstilbestrol (DES), dienestrol, trans-stilbene, progesterone, testosterone, 5 alpha-dihydrotestosterone, or olive oil for the first 5 days after birth. When the females were killed at 8 weeks after birth, all the estrogens, effective at different dose levels (10(-2) to 5 microgram/day), had resulted in the display by several of the cervicovaginal preparations studied of a heterotopic columnar epithelium (HCE) in regions where females given injections of olive oil, testosterone, 5 alpha-dihydrotestosterone, progesterone, or trans-stilbene had only the normal squamous epithelium. The further fate of the HCE was followed at two later age stages, 36 to 52 weeks and 14 to 17 months. The HCE developed into glandular-like structures penetrating into the stroma and justifying the designation of adenosis. DES resulted in a more pronounced adenosis than did 17 beta-estradiol; in both cases, metaplasia was a striking component of the adenosis regions. Development of adenosis from HCE was dependent upon presence of the ovaries. Some preparations from 44-week-old females given DES injections showed signs of a beginning malignant transformation in the adenosis regions, more evident in 17-month-old females. Among the 23 preparations in the latter group, 8 had changes morphologically indicating malignancy with examples of adenocarcinoma, mixed carcinoma, and squamous carcinoma. Because of the seemingly low aggressive nature of this malignancy, the term "pseudocarcinoma" is discussed. Ten- to 12-week-old BALB/c and C57BL/6 females given DES injections neonatally had HCE in the uterine cervix and vaginal fornices after neonatal DES injections. Differences in extension of HCE were observed after DES injections for three different 5-day periods in th neonatal and immature stages of NMRI females. An interaction between different DES-sensitive parameters to result in the pseudocarcinomas is discussed.

Natural killer cell activity and tumor susceptibility in female mice treated neonatally with diethylstilbestrol.

Female NMRI or AKR/J mice were given daily s.c. injections of 5 micrograms diethylstilbestrol (DES) in 0.025 ml olive oil, or of olive oil only, for the first 5 days after birth. At the age of 6 to 7 weeks, both DES-treated females and control females were killed, and the cytotoxic activity of the spleen cells against standard natural killer cell target YAC-1 cells as well as the natural killer cell-sensitive I-522 cells and relatively insensitive I-51 AKR lymphomas were tested. The cytotoxic activity against I-51 cells was similar for DES-treated and control females while the DES-treated females had only about one-half the cytotoxicity activity to I-522 and YAC-1 cells as did controls. Control females eliminated radioactivity derived from 125I-labeled YAC-1 and I-522 target cells injected i.v. faster than did DES-treated females, while the results were similar for both animal groups when using I-51 cells. The cumulative death incidence was higher for DES-treated females than for control females after incubation with low numbers of I-522 cells but similar for both groups when using I-51 cells. Finally, the incidence of females developing methylcholanthrene-induced sarcomas, using a simple low-dose injection (10 or 20 micrograms), was higher among DES-injected animals than among controls. Taken together, the results indicate that female mice treated neonatally with DES have a functionally defective natural killer cell population, resulting in increased tumor susceptibility.

Augmentation of natural killer cell activity after arterial embolization of renal carcinomas.

Preoperative embolization of the renal artery has been reported to improve the survival of patients with advanced renal carcinomas compared to operative treatment only. To investigate possible immunological consequences of tumor embolization, natural killer (NK) cell activity in peripheral blood was investigated immediately before and at different time intervals after occlusion of the renal artery by insertion of a metal coil. A slight increase in NK activity could be observed 24 hr postembolization while a marked augmentation was seen after 48 hr. The high NK activity persisted up to 96 hr after embolization, the last time period included in the study. Two patients undergoing the same procedure but in whom embolization was unsuccessful showed no alteration in NK activity. It is suggested that interferon produced by macrophages activated by the necrotizing tumor might be responsible for the augmentation of NK activity.

Antibody-targeted superantigens induce lysis of major histocompatibility complex class II-negative T-cell leukemia lines.

CTLs bearing certain T-cell receptor V beta-regions are directed by the bacterial superantigen Staphylococcus enterotoxin A (SEA) to lyse MHC class II-positive cells. In order to extend superantigen-dependent cytotoxicity to MHC class II-negative carcinoma cells, covalent conjugates of superantigen and mAbs against surface markers of these cells have been used. We now describe a novel strategy which allows rapid selection of mAb suitable for superantigen targeting against MHC class II-negative tumor cells. A recombinant fusion protein of protein A and SEA binding to the mAbs CD7 or CD38 was able to mediate T cell-dependent lysis of MHC class II-negative Molt-4 and CCRF-CEM acute lymphatic leukemia cell lines. Lysis was dose dependent and correlated with E:T cell ratio. In contrast, SEA alone did not induce any significant lysis. In order to decrease the MHC class II affinity of the protein A-SEA complex, a point mutation was introduced into SEA (protein A-SEA mu9). The mutated fusion protein had similar potency as protein A-SEA against Molt-4 cells but was 100-fold less active against MHC class II-positive cells. Considering the efficiency and specificity of the mutated SEA protein interacting with mAb in targeting T lymphocytes against MHC class II-negative leukemia cells while only marginally affecting normal MHC class II-positive cells, we suggest the development of SEA-mAb fusion proteins as a potential adjuvant therapy of leukemias.

Superantigen-based immunotherapy: a phase I trial of PNU-214565, a monoclonal antibody-staphylococcal enterotoxin A recombinant fusion protein, in advanced pancreatic and colorectal cancer.

PURPOSE: To establish the maximum-tolerated dose (MTD) and define the toxicities of a single-dose infusion of PNU-214565, a recombinant Escherichia coli-derived fusion protein of Staphylococcal enterotoxin A (SEA) and the Fab-fragment of the C242 monoclonal antibody in patients with advanced colorectal and pancreatic carcinomas. To investigate the capability of PNU-214565 to induce a superantigen (SAg) response resulting in cytokine production and tumor regression. PATIENTS AND METHODS: Twenty-one patients (age range, 39 to 76 years; median, 64; 12 men, nine women; 18 colorectal, three pancreatic cancers) were treated with a single 3-hour infusion of PNU-214565, with doses ranging from 0.01 to 1.5 ng/kg. All patients had prior chemotherapy and a good performance status Eastern Cooperative Oncology Group [ECOG] performance status [PS] = 0 [n = 10]; PS = 1 [n = 11]), 10 had prior radiation, and 18 had prior surgery. RESULTS: Fever and hypotension were the most common toxicities. Fever of any grade occurred in 16 of 21 patients (76%): four of 21 (19%) with grade 2 and two of 21 (9.5%) with grade 3. Hypotension of any grade occurred in 13 of 21 (62%): four of 21 with grade 2 and one of 21 (5%) with grade 3. Interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF alpha) induction correlated with toxicity. In the two patients with grade 3 fever, peak IL-2 and TNF alpha levels were 2.9 IU/mL and 165 pg/mL, and 8.3 IU/mL and 245 pg/mL, respectively. Transient, > or = 50% decreases in circulating monocytes were observed in 17 of 21 patients as early as 0.5 hours (median time, 2 hours) from the start of infusion. Decreases (mean 33%) in circulating lymphocytes were observed in seven of 21 patients. All three patients with grade 3 toxicity were treated at the 0.5-ng/kg dose. The significance of baseline anti-SEA, human antimouse antibody (HAMA), CA242-soluble antigen levels, and T-cell receptor variable beta region (TCR V beta) subsets and histocompatibility leukocyte antigen-DR (HLA-DR) genotypes was assessed as possible predictors of toxicity. All toxicities were transient and easily managed. No grade 3 toxicity occurred at the higher dose levels. CONCLUSION: PNU-214565, a SAg-based tumor targeted therapy, is safe when given as a single 3-hour infusion at doses up to 1.5 ng/kg. The MTD for a single dose was not determined. The safety of a repeated dose schedule is currently under investigation, beginning with doses determined to be safe in this trial.

T cell activation pathways: B7, LFA-3, and ICAM-1 shape unique T cell profiles.

Two signals are required for induction of cell proliferation and cytokine production in resting T cells. Occupancy of the T cell receptor by antigen/MHC complexes delivers the first signal to the T cell, while the second signal is provided by interaction with costimulatory ligands on APC. CD2, LFA-1, and CD28 are the major costimulatory and adhesive molecules on T cells and bind to the LFA-3, ICAM-1 and B7 ligands, respectively, on APC. LFA-3 plays a central role for naive and memory T helper cells during the early phase of an immune response. The LFA-3/CD2 pathway initiates strong antigen-independent cell adhesion, substantial expansion of naive T helper cells, and induction of large amounts of IFN-gamma in memory cells. The release of IFN-gamma may upregulate expression of ICAM-1 and B7 on APC and allows multiple adhesion pathways to amplify the immune response. The LFA-1/ICAM-1 pathway stimulates adhesion and cell proliferation more efficiently in memory T helper cells than in naive cells. Further, the results suggest that naive T helper cells express functionally inactive LFA-1 molecules on the cell surface, which may have a physiological role in keeping these cells in a resting state. B7 costimulation superinduces IL-2 production in both naive and memory T helper cells and generates long-lasting cell proliferation. This permits transition from an autocrine to a paracrine immune response. Coexpression of B7/LFA-3 provides an optimal APC function and enables a vigorous T cell response to minute amounts of antigen. AP-1 and NF-kappa B transcription factors are involved in the induction of several cytokine gene promoters and play a central role in the regulation of IL-2 gene transcription. LFA-3 costimulation only moderately enhances AP-1 DNA-binding activity and does not influence the NF-kappa B activity induced by TCR engagement, whereas B7 costimulation induces large amounts of NF-kappa B and AP-1 activity in T helper cells. The costimulatory ligands represent a family of adhesion molecules with considerable redundancy. Interfamily redundancy of LFA-3, B7, and ICAM ligands offers an opportunity to regulate distinct T cell response profiles in various microenvironments at separate time points of an immune response.

Antibodies are capable of directing superantigen-mediated T cell killing of chronic B lymphocytic leukemia cells.

The bacterial superantigen staphylococcal enterotoxin A (SEA) is a highly potent activator of cytotoxic T cells when presented on MHC class II molecules of target cells. Our earlier studies showed that such SEA-directed T cells efficiently killed chronic B lymphocytic leukemia (B-CLL) cells. With the ultimate goal to replace the natural specificity of SEA for MHC class II molecules with the specificity of a monoclonal antibody (mAb), we initially made a mutated protein A-SEA (PA-SEAm) fusion protein with > 100-fold reduced binding affinity for MHC class II compared to native SEA. The fusion protein was successfully used to direct T cells to B-CLL cells coated with different B lineage specific (CD19, CD20) or associated (CD37, CD40) mAbs. The PA-SEAm protein was 10-100-fold more potent against mAb coated compared to uncoated HLA class II+ B-CLL cells. No correlation was seen between the amount of mAb bound to the cell surface and sensitivity to lysis. Preactivation of B-CLL cells by phorbol ester increased their sensitivity, and lysis was dependent on ICAM-1 molecules. However, no preactivation of the target cells was needed when a cocktail of two or four mAbs was used. Circulating leukemia and spleen cells were equally well killed. We conclude that the natural target specificity of SEA, MHC class II, can be reduced by mutagenesis and novel binding specificity can be introduced by linkage to tumor reactive mAbs. Our findings encourage the construction of recombinant SEA mutant fusion proteins for specific T cell therapy of hematopoietic tumors such as B-CLL.

The content of a specific cell product in the vaginal epithelium of normal and neonatally estrogenized mice: its dependence on an estradiol-prolactin interaction.

The amount of an immunological marker (CVA) in mucified cells of the mouse vaginal epithelium was quantified by a mixed hemagglutination technique for tissue sections. Immature mice, adult mice which had been estrogenized neonatally (5 mug diethylstilbestrol daily for the first five days after birth), and adult non-estrogenized mice were studied. All adult animals were castrated 7-10 days before starting the experiments. Injections of 5 mug estradiol-17beta (48 and 24 h before killing the animals) increased the amount of CVA in all three groups of animals, but most markedly in the neonatally estrogenized mice. The amount of CVA found following estradiol treatment was decreased in adult animals injected with the ergot alkaloid CB154 (0.5 mg twice daily for 6 days) in addition to the hormone. This partial block of the estradiol-induced CVA response by CB154 was relieved by exogenous rat prolactin. The CVA content in immature animals was not influenced by CB154, given alone or together with estradiol. Combined treatment with estradiol and rat prolactin (3 mug every 8 h for 6 days) increased more efficiently than estradiol alone the amount of CVA in immature and adult nonestrogenized animals. Prolactin injected alone had no effect on the CVA content. These data strongly suggest a synergistic action of estradiol and prolactin in augmenting the epithelial CVA content. Explants of the vaginal wall from normal and neonatally estrogenized mice were grafted into the thigh muscles of newborn mice, every host carrying one graft from both types of animals. The CVA content in the epithelium of the two grafts increased to the same level in response to estradiol. When the hosts were injected with estradiol and prolactin, the CVA content was higher in grafts from estrogenized donors than in those from nonestrogenized animals. Our results demonstrate that the mucified vaginal cells in adult, neonatally estrogenized mice have a content of CVA which is higher than in nonestrogenized animals. This difference may be ascribed to hormonal factors (estradiol-prolactin) as well as to persistent effects in the vaginal cells as a result of the neonatal estrogen treatment.

A simple and rapid method to distinguish between rosettes and non-specific aggregation of erythrocytes in the rosette assay for human T-lymphocytes.

A method is described for rapid and simple visualization of lymphocytes in the rosette assay for human T-lymphocytes. A drop of acridine orange (pH 7.4) is added to the suspension of rosetted cells and allowed to incubate for 1 min on ice. Nucleic acids are then visualized in a fluorescence microscope by excitation at 430-500 micrometer. Simultaneous semi-illumination with light microscopy allows enumeration of total lymphocytes and the number of lymphocytes rosetted with sheep red blood cells. The central lymphocyte is clearly distinguishable even when it is crowded with red cells, and this makes the differentiation between rosettes and non-specific aggregation of cells easy.

Efficient selection of scFv antibody phage by adsorption to in situ expressed antigens in tissue sections.

The present report describes the development and application of an efficient method for the direct adsorption/selection of antibody phage using antigens expressed in situ in cryostat tissue sections. In a model system, scFv phage directed towards an epitope on the GA733-2 epithelial glycoprotein expressed in colorectal carcinoma tissue could be specifically enriched up to 1500 fold in single-pass experiments and a million fold after three rounds of selection. Enrichment efficacy was directly proportional to the fraction of antigen positive area over the total area. Sufficient enrichment was achieved at an area fraction of less than four percent, thereby permitting the selection of antibodies to sub-populations of cells or to tissue sub-structures. The general usefulness of the method was demonstrated when a combinatorial scFv antibody phage library derived from melanoma immunized non-human primates was selected in tissue sections of metastatic melanoma. Individual scFv antibodies from enriched phage populations demonstrated different binding specificities, reflected in extracellular and cellular tissue staining patterns which included tumor cell surface reactivity. This method should be particularly useful for the identification of antigens which are only expressed during specific in vivo conditions, and overcomes a major limitation of currently used selection protocols.

Immunomodulation of autoimmunity by linomide: inhibition of antigen presentation through down regulation of macrophage activity in the model of experimental autoimmune encephalomyelitis.

Linomide (quinoline-3-carboxamide, LS-2616), a synthetic immunomodulator, protects animals against a variety of experimental autoimmune diseases. In experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis (MS), linomide blocks both the clinical and histological signs of the disease, without inducing generalized immunosuppression. In the first clinical trial in patients with MS, linomide was shown to inhibit the progression of the disease. In the present study we investigated several aspects of the mechanisms of action of this immunomodulator. We found that linomide can inhibit acute EAE even when given as pretreatment, prior to induction of disease (days - 10 to 0). This inhibitory effect was reversed by adoptive transfer of naive spleen cells. A short course (7 days) of linomide treatment also inhibited EAE, especially when administered immediately after disease induction. Spleen cells from linomide-treated mice failed to present myelin antigens to T-cell lines in vitro. The defective antigen presentation was normalized by anti-oxidants such as 2-mercaptoethanol. The proportion of Mac1+ cells in the spleens of linomide-treated mice was significantly reduced and macrophage growth was inhibited in long term cultures of spleen cells derived from linomide-treated animals. Our findings suggest that the effect of linomide on EAE may be attributed, at least in part, to inactivation of antigen presenting cells, possibly following a short period of over-stimulation and increased oxidant production. This mechanism may play a universal role in the regulation of autoimmune reactivity and merits further investigation.

Functionally involved cell surface antigens on murine lymphokine activated killer cells.

To investigate the possible involvement of some cell surface structures on lymphoid cells in the functional activity of lymphokine activated killer (LAK) cells, a number of monoclonal antibodies (Mab) against such structures was studied for their ability to inhibit LAK activity in a standard cytotoxicity assay against the natural killer-insensitive target cell EL-4. Almost complete inhibition of LAK activity resulted from incubation with antibodies to the LFA-1 antigen, while blocking of the Lyt 2 antigen reduced cytotoxic activity about 50%. Mab to T-200 gave a weak and inconsistent inhibitory activity, while antibodies to Thy 1, L3T4, IL-2 receptor and MHC class I antigens were without effect. Mab to LFA-1 and Lyt 2 inhibited LAK activity towards EL-4, YAC-1 and differentiated F-9 teratocarcinoma cells, but did not affect LAK-mediated killing of undifferentiated F-9 cells. Experiments with separate preincubation of effector and target cells revealed that both LFA-1 and Lyt 2 inhibited LAK activity at the effector cell level only.

Locally superantigen-activated peritoneal cytolytic T lymphocytes belong to the CD8+ CD45RC- subset and lyse MHC class II+ tumor cells.

Bacterial encoded superantigens (SA) are capable of activating and targeting cytolytic human and mouse T lymphocytes (CTL) to lyse major histocompatibility complex class II positive (MHC class II+) target cells. In this study both in vitro and in vivo activated rat CTL were directed against MHC II+ tumor targets by bacterial encoded SA. Polyclonal in vitro activation of rat peripheral blood T lymphocytes generated CTL capable of killing MHC class II+ human BSM cells coated by staphylococcal enterotoxin (SE) -A, -E, -D, and TSST-1 but not by SEB or SEC1-3. Allo selective peritoneal CTL generated by intraperitoneal stimulation with allogeneic spleen cells were directed against BSM cells by SEA, -D, and -E but not by SEB, SEC1-3 or TSST-1. Based on the above observations, and in order to locally activate CTL, SEA was chosen for in vivo priming of rats by intraperitoneal inoculation of the toxin. SEA injection generated highly cytolytic CTL, and maximum cytolytic responses were seen at 50-250 micrograms SEA per animal with a peak in response 48-72 hours after injection of the toxin. The cytolytic activity of peritoneal SEA reactive effector cells was confined to the TCR alpha beta+ CD4- CD8+ CD45RC- cell population. MHC class II- colon carcinoma cells were insensitive to lysis by SEA reactive CTL but colon carcinoma cells induced to express MHC class II by interferon-gamma (IFN-gamma) treatment were efficiently lysed in the presence of SEA. Comparison of rat and human MHC II+ colon carcinomas revealed a peak in sensitivity to lysis at 10-100 ng SEA/ml for both tumor targets. These findings suggest that superantigens can be used in local immunotherapy of peritoneal tumors such as ovarian and colorectal carcinomatosis, with inducible or constitutive expression of MHC class II.

Delayed hypersensitivity response to oxazolone in neonatally estrogenized mice.

Neonatal NMRI mice were treated with diethylstilbestrol (DES) fof the first 5 days after birth. At 6 and 9 months the same animals were tested for delayed hypersensitivity reaction using an ear test with oxazolone. The DES treated animals had a diminished response to oxazolone compared with controls.