Hilliard Festenstein

A new sensitive assay for antibody against cell surface antigens based on inhibition of cell-dependent antibody-mediated cytotoxicity: II. Mechanism

Abstract The underlying mechanism of the cytotoxicity inhibition assay (CIA) described in an earlier report was investigated. Inhibition of the cell-dependent lysis of antibody-coated target cells by antibody was dependent (i) onspecific binding of the antibody to some cells in the effector cell population, (ii) on the presence of the F c portions of the antibody molecules, and (iii) on the amount of anti-target cell antibody in the system. The inhibitory component, which was not present in the supernatants, could be shown to represent antibody-cell complexes. On the basis of these findings, we proposed for the mechanism of the CIA a competition on the level of the F c receptor of cytotoxic effector cells between target cell-bound antibody and any other antibody-cell complex in the incubation mixture. Further information about the nature of the inhibitory complexes was obtained from experiments where antibody-coated “third party” cells were treated with complement. The resultant complexes of dead cell membranes, antibody, and complement appeared to have inhibitory potency similar to that of the antibody-coated live cells. Interpretations and implications of these results were discussed.

Inhibition of cell-dependent cytotoxicity as an assay for mouse alloantibody.

MANY assays for alloantibody depend on functional activities of the Fc portion of the immunoglobulin molecule. These functional activities are acquired when the antibody binds to its target cell. Complement binding, as demonstrated by lysis or complement fixation, has been the most widely used of these activities. In man, alloantibody can also be detected by its ability to mediate cell-dependent lysis of target cells1–3. In this system, effector cells (human peripheral blood leukocytes) lyse allogeneic chromium 51Cr-labelled target cells (lymphocytes) in the presence of the appropriate alloantibody, providing a sensitive, complement-independent assay for alloantibody. In contrast, we have found that mouse alloantisera do not usually mediate lysis of mouse target cells (lymph node lymphocytes, phytohaemagglutinin (PHA) blast cells, and tumour cells) by mouse effector cells (spleen cells). Data supporting this conclusion will be presented elsewhere.

Human Cytotoxic T-Cells against Measles Virus-Infected and Myelin Basic Protein-Coated Targets Are Cross-Reactive

Cytotoxic T lymphocytes (CTL) which recognized measles virus antigens were generated by in vitro sensitization of peripheral blood lymphocytes from normal volunteers against autologous measles virus-infected lymphocytes. Cytotoxicity of measles virus-infected targets by these effectors was considerably enhanced when the effector-target cell mixtures were incubated in presence of 10 or 100 ng myelin basic protein (MBP) for the 3-hour duration of the 51Cr release assay. In most experiments, specific release of radioisotope was doubled or tripled. Bovine serum albumin caused only slight increases in cytotoxicity. The killing of allogeneic target cells by alloimmune CTL was not affected by either of these reagents. Measles-specific CTL were also able to kill target cells that were cultured overnight in presence of MBP but washed prior to the assay. Conversely, CTL generated by culturing lymphocytes in presence of MBP for 6 days were able to kill MBP-coated and measles virus-infected target cells. The implications of these findings in the pathogenesis of multiple sclerosis are discussed.

Structural variations in the H-2 genes of AKR lymphomas.

K36.16 is an AKR H‐2k thymoma which expresses an aberrant H‐2Dd‐like allospecificity, does not have a detectable amount of the H‐2Kk syngeneic antigen and grows very easily in syngeneic mice. By DNA‐mediated gene transfer experiments, we were able to obtain transformed clones which do express the H‐2Kk molecules and are rejected by AKR mice. Southern hybridization was performed to assess whether any gross changes had occurred in the K36.16 H‐2K locus or elsewhere in the MHC, which might explain the lack of H‐2K expression and/or the presence of the aberrant H‐2Dd‐like allospecificity. Specific H‐2 class I DNA probes were used to compare the K36.16 genomic DNA with normal AKR thymus DNA after digestion with a variety of restriction enzymes. After hybridization with the pH‐2IIa probe a 2.8 kb ‘Hind III’ fragment was identified in the K36.16 genomic DNA which is absent from AKR DNA. The pH‐2IIa probe detects the third, transmembrane and cytoplasmic domains of class I genes. Although these changes are indicative of MHC genome modifications it is not yet possible to link these specific Southern blot pattern variations with the phenotypic changes mentioned above.

Mechanisms of “cytostasis” of tumours in vitro by syngeneic lymphoid cells of tumour bearers

Abstract The cytostasis assay is an in vivo-in vitro radioactive technique which detects antitumour responses of the syngeneic tumour-bearing hosts. Examination and characterization of effector mechanisms at the cellular and humoral levels revealed that the cytostasis assay using Meth A (a 3-methylcholanthrene-induced) tumour was T cell independent. Furthermore, both B cells and macrophages were required. It was concluded that the mechanism involved complement-dependent antibody-mediated lysis of the tumour cells, with B cells producing antibody and macrophages producing the complement components during incubation. However, antibody-dependent cell-mediated cytotoxicity with or without complement could not be completely excluded. Although antibody was detected in vivo , specific antibody against Meth A tumour was produced in vitro by cultured lymphoid cells from the tumour-bearers. Antibody-coated Meth A cells caused regression of some tumours when inoculated into BALB/c mice. When these regressor mice were rechallenged with tumour, they were found to be permanently immune to the tumour. In the light of these findings, the role of antibody in the protection of tumours and its implications are discussed.

PROTECTION OF NEWBORN MICE FROM GRAFT VERSUS HOST DISEASE BY MATERNAL PRE‐IMMUNIZATION

Alloimmunization of BALB/c (H‐2d) female mice with allogeneic spleen cells from C57BL/6 (H‐2b) or CBA/H (H‐2k) mice protects BALB/c offspring from graft‐versus‐host disease (GVH‐D) following neonatal intraperitoneal inoculation of high doses of spleen cells respectively of C57BL/6 or CBA/H strains of mice.

An RFLP Study of HLA-D Gene Polymorphism in Multiple Sclerosis

An association between the demyelinating central nervous system disease, multiple sclerosis (MS), and DR2, DQw1 has been defined in numerous Northern European populations (1,2). The relative risk conferred by possession of DR2 is low, ranging from 2.0 to 3.8 in different studies (3). This study was designed to explore an association between HLA-D-region alleles and MS in more detail, using restriction fragment length polymorphism (RFLP) analysis. The selection of enzyme/probe combinations was based on a recent RFLP study conducted in our laboratory in which we used the pooled DNA approach first described by Arnheim et al. (4). Using pooled DNA samples from DR2+ patients, DR2+ controls, DR2− patients, and DR2− controls, we were able to screen 70 enzyme/probe combinations including DRβ, DQβ, DPβ, DQα, and DPα and 14 different restriction enzymes. This analysis highlighted MspI/DQα fragments that occurred at a higher frequency in patients than controls, and provided the basis for a multicenter study.