Claudia Henry

Directed fusion in hybridoma production

We have attempted to increase the frequency of azobenzene arsonate-specific hybrids by bridging specific B cells to the myeloma partner cells prior to polyethylene glycol-induced fusion. Bridging was accomplished by prelabeling the B cells with avidin-labeled antigen and incubating them with myeloma cells that had been modified directly with biotin. We have tested this system of hybridization with B cells from normal mice, and mice undergoing both primary and secondary responses. We found that the method is fruitful for IgG-secreting hybridomas of moderately high affinity.

In vitro anti-hapten response to a lactoside-conjugated protein

Abstract This paper describes the in vitro response of mouse lymphocytes to the hapten, azophenyl lactoside (Lac), coupled to the protein carrier, keyhole limpet hemocyanin, (KLH), and attempts to characterize and quantify the specific B and T cells involved. The system studied in detail has used spleen suspensions from mice which received Lac-KLH in complete adjuvant some months previously and a subsequent iv injection. The mean IgG plaque response of such cultures was about 15,000 with a corresponding IgM response about 5% of that value, similar in magnitude and Ig class distribution to in vivo responses. Primary responses were small, reflecting the low frequency of Lac precursors in normal mice. From dilution studies, we have estimated that the frequency of precursors specific for the Lac epitope in primed and normal mice is of the order of 10 −5 and 10 −7 , respectively. The clonal yield of plaques from a stimulated B cell was about 150, independent of T cell concentration. The Lac response was dependent on the presence of adherent cells. It also shows a stringent requirement for carrier-specific T cells, which could not be satisfied by nonspecific T cell products or the addition of B cell mitogens. We have found that both the IgM and the IgG Lac responses are dependent on specific T cell help.

Fluorescence visualization of Ia antigens on T cells

Abstract Visualization with the fluorescence microscope of Ia antigens on T cells has previously not been successful. In this paper we have used fluorescence hapten-sandwich labeling to examine cells for the simultaneous display of Ia antigens and T-cell-specific antigens. In contrast to small unstimulated T cells which bear only small amounts of Ia antigens, we find that concanavalin A-stimulated T-cell blasts exhibit amounts that are clearly recognizable. The predominant specificity expressed is encoded by the I -A subregion and appears to be indistinguishable from that on B cells. Smaller subpopulations exhibit products coded by the I -C and I -J subregions. Separation of subpopulations of T cells on the basis of selective display of Ia antigens appears only to be feasible with stimulated cells.

Converting Sendai virus into a specific fusogen whose cell target can be selected

Covalent intermolecular hybrids of Fab anti-hemagglutinin-neuraminidase (HN) monoclonal antibody and avidin were prepared and characterized. These conjugates were used to block and redirect the fusion activity of Sendai virus (SV). After incubation of SV with Fab anti-HN: avidin conjugate on ice for 1-2 h, the SV fused only those P815 or BW5147 cells which were labeled with biotin-modified anti-cell surface immunoglobulin. The levels of cell-cell fusion obtained were at least as high as those achieved with unmodified SV and unlabeled P815 or BW5147 cells. These results demonstrate that it is possible to block the normal agglutinating activity of the HN molecules of SV and to introduce a new cell recognition feature without negating the fusogenic potential of the virus. Such an approach may be useful in harnessing the fusion activity of SV to a targeted delivery system for microinjection of macromolecules into selected cell populations.

On the precision of the plaque count

Abstract A simple method of measuring “procedural error” of plaque counts of antibody-secreting cells which affects counts in addition to the Poissonian error inherent in counting is proposed and evaluated. The two components are easily combined to give an expression for the variance of a single count. We have examined two sets of duplicate counts of antihapten plaques, the first scored by a modification of the original method, and the second by the original procedure, and have found them to be comparable in precision. We have reexamined published data of an earlier analysis of anti-red cell plaques and the half-leaf method of assaying plant viruses. The procedural error for the former was slightly larger and for the latter 60 times as large as for our data sets. Since procedural error is a function both of the method and the skill of those handling it, we recommend its estimation for individual laboratories, and have outlined a simple method that achieves this end without special effort. The knowledge of procedural error, incorporated into a formula for the variance of a single count, leads to a considerable long-term improvement in the economy of experimentation involving counts. Instead of placing confidence in fairly unstable variance estimates from small numbers of replicates in a given experiment, one may rely on documented experience of relevant history. In practical terms, a simple z-test statistic can be computed which allows one to judge differences between a single “control” and a single “treatment” count.

ON THE ROLE OF Ia IN B-T CELL INTERACTION

The anti-Lac B precursor cells from Balb/c mice which survive cytotoxic treatment with anti-Ia k will respond to Lac-KLH in culture but require more KLH helper T cells than the untreated B cell population. These findings are not explainable by the simple Poisson assumption of a constant target of T-B interaction. We propose an interaction theory with variable Ia target on the B cell surface. The theory quantitatively predicts the observed dose-response relationships, and implies a central role for Ia in T-B interaction.