Cytotoxic T lymphocyte recognition of the influenza hemagglutinin gene product expressed by DNA-mediated gene transfer.

We have used the technique of DNA-mediated gene transfer to examine cytotoxic T lymphocyte (CTL) recognition of the product of the cloned A/JAPAN/305/57 hemagglutinin (HA) gene in murine (L929) cells. Using both heterogeneous and homogeneous (clonal) populations of type A influenza-specific CTL, we have demonstrated that the HA molecule can serve as a target antigen for both the subtype-specific and the cross-reactive subpopulations of influenza-specific CTL. Our results also raise the possibility that other virus-specified polypeptides may serve as target molecules for cross-reactive CTL.

Expression of a murine polyclonal T cell receptor marker correlates with the use of specific members of the V beta 8 gene segment subfamily.

A series of murine T lymphocyte clones were examined for reactivity with the KJ16-133 and F23.1 mAbs. Clones that were KJ16-133+,F23.1+ and KJ16-133-,F23.1+ were identified, but no KJ16-133+,F23.1- clones were observed. Within our panel of clones, therefore, the KJ16-133 antibody identifies a subset of F23.1+ cells. All F23.1+ clones examined express members of the V beta 8 subfamily of beta chain variable region genes; clones expressing V beta 8.1 or V beta 8.2 reacted with both KJ16-133 and F23.1, while clones expressing V beta 8.3 reacted only with F23.1. Thus, the differential reactivity of the KJ16-133 and F23.1 antibodies with cloned T cells correlates with the V beta gene expression of each clone. Reactivity with these antibodies should therefore be of utility for predicting the V beta gene expression in some T cell clones.

Folate analogues altered in the C9-N10 bridge region. 18. Synthesis and antitumor evaluation of 11-oxahomoaminopterin and related compounds.

The chemical synthesis of 11-oxahomoaminopterin (1) has been carried out using procedures which were also found to be applicable to the synthesis of 11-oxahomofolic acid (2). Reaction of 1-bromo-4-[p-(caarbomethoxy)phenoxy]-2-butanone (10) with sodium azide gave 1-azido-4-[p-(carbomethoxy)phenoxy]-2-butanone (11). Protection of the carbonyl group of 11 as the ethylene ketal and subsequent base hydrolysis of the product gave 1-azido-4-(p-carboxyphenoxy)-2-butanone ketal (13). The glutamate conjugate 14 was prepared from 13 by the isobutyl chloroformate method and was hydrogenated to diethyl N-[(alpha-amino-2-oxo-4-butanoyl)-p-anisoyl]-L-glutamate ketal (15). Reaction of 15 with 6-chloro-2,4-diamino-5-nitropyrimidine (16) and 2-amino-6-chloro-4-hydroxy-5-nitropyrimidine (17) and deprotection of the corresponding products gave the intermediates 18 and 19, which were elaborated to 1 and 2 using a series of steps involving deprotection, dithionite reduction, cyclization, oxidation, and hydrolysis. Although 11-oxahomoaminopterin showed antifolate activity against two folate-requiring microorganisms and inhibited Lactobacillus casei DHFR, it was inactive against L-1210 leukemia in mice at a maximum dose of 48 mg/kg. Compound Lactobacillus casei DHFR, it was inactive against L-1210 leukemia in mice at a maximum dose of 48 mg/kg. Compound 1 was also tested for its ability to be transported via the methotrexate transport system using the L-1210 and Ehrlich tumor cell lines, and these results are compared with those of related analogues. The growth inhibitory activity of 1 in the L-1210 cell lines in culture was found to be 15 times weaker than that of methotrexate.

Anti-T cell receptor antibodies fail to inhibit specific lysis by CTL clones but activate lytic activity for irrelevant targets.

In this report, we describe the functional effects of anti-T cell receptor antibodies on a panel of MHC-restricted, influenza virus-specific CTL clones. Approximately 25 to 30% of these clones are recognized by KJ16-133, an anti-T cell receptor monoclonal antibody presumably specific for products of the V beta 8 gene family, and an antibody with similar specificity, F23.1. In contrast to most previous reports, both KJ16-133 and F23.1, over a wide range of antibody concentrations, fail to inhibit the antigen-specific effector function of these CTL. Instead, the antibodies activate the CTL to kill without regard for the MHC haplotype of the target cells or the presence of the appropriate viral antigen. This anti-T cell receptor antibody-induced cytolysis by our clones does not appear to be mediated by Fc receptors on target cells. Nuclear destruction of target cells as a result of antibody-induced lysis suggests that it occurs via a mechanism similar to antigen-specific lysis by CTL. In addition, both soluble bivalent F23.1 and F23.1 coupled-Sepharose beads are able to induce the secretion of interferon-gamma from these CTL clones.

Fine specificity and antigen receptor expression among influenza virus-specific cytolytic T lymphocyte clones.

Influenza virus stimulates a vigorous cytolytic T lymphocyte (CTL) response in the mouse that is directed to several virion polypeptides. This report examines the fine specificity of a panel of murine influenza-specific CTL clones restricted by MHC class I products of the H-2d haplotype. Ten of 22 A/JAPAN/305/57-specific CTL clones analyzed were directed to the A/JAPAN/305/57 hemagglutinin protein as detected by using target cells infected with a recombinant vaccinia virus containing hemagglutinin gene. Based on their fine specificity of hemagglutinin recognition, these clones defined four functional epitopes on the hemagglutinin. The remaining 12 cytolytic clones exhibited cross-reactivity for type A influenza viruses of the major human subtypes, and approximately 60% of these clones were directed to the nucleocapsid protein. KJ16-133 monoclonal antibody analysis of the utilization of the T cell receptor V beta 8 gene segment subfamily revealed that members of this V beta gene subfamily are expressed by both hemagglutinin- and nucleocapsid-specific MHC class I-restricted CTL (and by influenza-specific MHC class II-restricted T lymphocytes as well). These results suggest that CTL detect several distinct antigenic sites on the hemagglutinin. In addition, these results reveal no direct correlation between viral antigenic specificity and V beta gene expression by these virus-specific CLT clones.

Stable expression of cytolytic activity by influenza virus-specific cytotoxic T lymphocytes.

Influenza-specific immune cytotoxic T lymphocyte (CTL) populations maintain a constant level of in vitro cytolytic activity. This is demonstrable with both heterogeneous populations of anti-viral CTL from immune donors and long-term CTL clones derived from primed CTL precursors. Cytolytic machinery is stably expressed by these CTL populations under a variety of in vitro cultivation conditions. This finding is in contrast to results with alloreactive CTL generated by stimulation of primed CTL precursors that lose cytolytic activity on a per cell basis with time after stimulation. The results indicate that virus-specific, cloned CTL that stably express cytolytic activity are representative of the heterogeneous populations from which they are derived and further suggest a qualitative difference in the regulation and expression of cytolytic machinery between heterogeneous populations of influenza-specific CTL and alloreactive CTL.