THE IMMUNOGENICITY OF ANTIGEN BOUND TO THE PLASMA MEMBRANE OF MACROPHAGES

Macrophages were cultured for several hours after a brief exposure to radio-iodinated keyhole limpet hemocyanin. Most of the hemocyanin taken up by the macrophages was rapidly catabolized and eliminated from the cell. A few molecules were retained on the plasma membrane of the cells for prolonged periods and were not subject to endocytosis and catabolism. These few molecules of hemocyanin bound to the plasma membrane were identified by observing the fixation of antibody fragments to macrophages at low temperature. The membrane-bound antigen, which could be removed by trypsin or EDTA, was of large molecular size, though heterogeneous. A great part of the immune responses of mice to hemocyanin bound to live macrophages could be abrogated by treatment of the macrophages in vitro with antibody or trypsin. Hence, most of the immunogenicity of hemocyanin bound to macrophages was attributed to the few molecules of antigen bound to the plasma membrane

Monitoring tumor antigen specific T-cell responses in cancer patients and phase I clinical trials of peptide-based vaccination

Numerous phase I and II clinical trials testing the safety and immunogenicity of various peptide vaccine formulations based on CTL-defined tumor antigens in cancer patients have been reported during the last 7years. While specific T-cell responses can be detected in a variable fraction of immunized patients, an even smaller but significant fraction of these patients have objective tumor responses. Efficient therapeutic vaccination should aim at boosting naturally occurring antitumor T- and B-cell responses and at sustaining a large number of tumor antigen specific and fully functional effector T cells at tumor sites. Recent progress in our ability to quantitatively and qualitatively monitor tumor antigen specific CD8 T-cell responses will greatly help in making rapid progress in this fiel

AN APPROACH TO THE QUANTITATION OF IMMUNOGENIC ANTIGEN

Using passively administered isotope-labeled anti-KLH to suppress the antibody response of rabbits to KLH, we have attempted to estimate the amount of antigen actually involved in stimulating antibody formation. Single and paired label tracer studies of passively administered anti-KLH IgG indicated that from 0.7 to 2.9 µg were utilized or involved by the antigen in the course of a 90% suppression of the response to 2 mg KLH. Tracer studies of labeled anti-KLH F(ab')2 fragments revealed the retention of from 2 to 3 µg of these fragments in the entire rabbit during a 60% suppression of the response to 1 mg KLH. Based on previously determined ratios of mixtures of KLH and suppressive amounts of anti-KLH in adjuvant, the antibody utilization data were converted to the probable amount of antigen or immunogen involved. It appears that after an injection of 2 mg of KLH approximately 0.2–0.5 µg of antigen persisted and reacted with antibody given 24 hr later. Since all of this persisting, reactive antigen may not be immunogenic, the above estimate of immunogen is probably high, but may serve to establish upper limits for the amounts of immunogen involved in stimulating antibody formation and provide a meaningful frame of reference for antigen tracer studies

GENERATION OF CYTOTOXIC T LYMPHOCYTES IN VITRO : III. Velocity Sedimentation Studies of the Differentiation and Fate of Effector Cells in Long-Term Mixed Leukocyte Cultures

Separation of cells by velocity sedimentation at unit gravity was utilized to investigate the physical properties of cytotoxic thymus-derived lymphocytes (CTL) generated in long-term mixed leukocyte cultures (MLC). In kinetic studies, CTL were found almost exclusively in the large cell fractions at the peak of the response on day 4, whereas the majority of CTL in day 14 MLC had the sedimentation properties of small lymphocytes. Reculture until day 14 of cells fractionated on the basis of size on day 4 indicated that the small CTL were derived exclusively from cells which had been large on day 4. Re-exposure of day 14 MLC cells to the original stimulating alloantigens resulted in significant cell proliferation and rapid regeneration of CTL activity. Cell fractionation experiments demonstrated that the cells in the day 14 MLC population which responded to the secondary allogeneic stimulus were small T lymphocytes, and that these cells rapidly developed into large, highly cytotoxic CTL following stimulation. Moreover, by restimulating on day 14 fractions which were selected on the basis of size on day 4, it was found that the responding small lymphocytes were themselves the progeny of cells which were large at the peak of the response. Since CTL and CTL progenitors showed concomitant changes in physical properties with time, the possibility exists that they belong to the same cell lineage, and hence that CTL can differentiate into cells which are no longer cytotoxic, but capable of mounting an anamnestic response

THYMUS-DERIVED (T) CELL IMMUNOGLOBULINS : PRESENCE OF A RECEPTOR SITE FOR IGG AND ABSENCE OF LARGE AMOUNTS OF "BURIED" IG DETERMINANTS ON T CELLS

Quantitation of surface and total cell Ig obtained after lysis by detergent, urea-acid treatment, and freeze-thawing were determined on spleen cells, thymus cells, and spleen cells specifically depleted of B cells. A two- to four-fold increase in measurable Ig was found after cell lysis. All cell populations showed a similar increase in measurable Ig indicating that no discordantly large amounts of buried Ig determinants were associated with the surface of T cells. The lack of appreciable amounts of T cell Ig was confirmed by immunoprecipitation of radioiodinated cells. A theta-positive lymphoma was described which, when grown in culture, lacked detectable surface Ig but contained a receptor site for IgG. This resulted in appreciable amounts of surface IgG being associated with the tumor line when isolated from ascitic fluid of tumor-bearing mice or after preincubation of cultured cells with either heat-aggregated IgG or normal mouse serum

COMPARISON OF THE IMMUNE RESPONSIVENESS OF NZB AND NZB x NZW F1 HYBRID MICE WITH THAT OF OTHER STRAINS OF MICE

The immune responsiveness of (NZB x NZW) F1 hybrid mice (NZB/W) has been compared with that of three other strains of mice, A/J, BALB/c, and CBA/J. The antigens used included sheep red blood cells (SRBC), keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), and human γ-globulin (HGG). It was found that important strain differences existed in the amount of antibody produced, but the relative immune responsiveness depended very much upon the nature of antigen. By comparison with the other strains tested, NZB/W mice had a higher antibody production to some antigens (SRBC and BSA) but were low responders to others (KLH). Induction of unresponsiveness to HGG by treatment with ultracentrifuged HGG was studied in the strains cited above. NZB/W mice became tolerant after injection of HGG ultracentrifuged at 100,000 g for 2 hr. Similar experiments carried out with another preparation of HGG (centrifuged at 20,000 g for 30 min) failed to reveal any abnormal behavior of NZB/W mice as compared to BALB/c or A/J mice. These results do not support the concept that NZB/W mice possess a general immune hyperreactivity or a relative inability to be made tolerant to protein antigens. However, they do not rule out the possibility that these mice have a genetically determined hyperresponsiveness to some antigens, in particular to nuclear antigens