Ralph M. Bernstein

HEAVY-CHAIN VARIABLE REGIONS IN CARCHARHINE SHARKS : DEVELOPMENT OF A COMPREHENSIVE MODEL FOR THE EVOLUTION OF VH DOMAINS AMONG THE GNATHANSTOMES

We determined the sequence of 18 DNA clones encoding VH regions of sandbar shark and bull shark. All of these sequences exhibit key structural coding features characteristic of known VH genes of higher vertebrates. These VH sequences disclosed considerable diversity, and can be divided into six families according to the criterion of 80% DNA sequence identity. The overlapping of some VH gene clones to two or more families is a particular feature found in carcharhine sharks, which suggests that VH diversification is a continuing process. The basic sequence patterns of heavy‐chain V regions found in all representative gnathanstomes and in VH of the shark heavy immunoglobulin igW provides evidence for selection of canonical residues in all VH structures, Elasmobranch VH sequences can be divided into two classes or clans, one comprising the ‘classical’ VH set and the other comprising VHs related to those of IgW (Vω). Phylogenetic analyses place the VH cluster as the root of all the classic VHs and indicates that the Vω set is most probably that of the primordial heavy chain.

Interferon γ (IFNγ) gene transfer of an EMT6 tumor that is poorly responsive to IFNγ stimulation: increase in tumor immunogenicity is accompanied by induction of a mouse class II transactivator and class II MHC

Abstract Interferon γ (IFNγ) is an important cytokine with immunomodulatory properties that include activation of immune cells and induction of class I and class II major histocompatibility complex antigens. In this study a retroviral vector was used to introduce the IFNγ gene into EMT6 tumor cells to assess the effect of IFNγ gene expression on tumor immunogenicity. Transfectants were selected in G418-containing tissue-culture medium and were determined to express the inserted IFNγ gene by reverse transcriptase/polymerase chain reaction. Flow-cytometric analysis revealed that parental unmodified EMT6 cells constitutively expressed only class I MHC and were poorly responsive to exogenous IFNγ stimulation, whereas class II MHC was induced in IFNγ-transfected cells. The induction of class II MHC in IFNγ-transfected cells correlated with the expression of a mouse class II transactivator gene that was dormant in unmodified or mock-transfected cells. In addition, IFNγ-gene-transfected tumor cells were found to secrete up to 17 ng IFN (equivalent to 75 units/106 cells) by enzyme-linked immunosorbent assay (ELISA). Whereas parental EMT6 cells grew unchecked, the growth of genetically modified tumor cells was significantly inhibited in immunocompetent mice. Rechallenge of animals that rejected an IFNγ-transfected EMT6 clone (EMT6-B17) with parental EMT6 cells resulted in tumor rejection, suggesting that IFNγ-transfected EMT6 cells were able to induce long-term immunity. Mixing experiments using gene-transfected and unmodified tumor cells demonstrated that 10% of IFNγ-transfected cells in the population was sufficient to protect mice against subsequent challenge with tumorigenic EMT6 cells. These studies demonstrate that the immunogenicity of tumor cells that are poorly responsive to exogenous IFNγ can be enhanced by inserting and expressing the IFNγ transgene. These findings also suggest a role for class II MHC in reducing tumorigenicity of the EMT6 tumor and inducing long-term tumor immunity.