Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy.

Carrier effect of concanavalin A-reactive and -non-reactive material in tuberculin PPD.

Tuberculin purified protein derivative (PPD) is a very potent T-cell reactive material in tuberculin-positive individuals, but the components responsible for this reactivity have not been adequately defined. Three purified mycobacterial proteins (MPB70, the BCG 65-kDa protein, and BCG antigen 85B) with different degrees of temperature sensitivity were iodine-labelled and added to BCG culture fluid, and the mixtures were autoclaved at 120 degrees C for 30 min to simulate the initial heating procedure used to prepare PPD. SDS-PAGE followed by protein staining and autoradiography showed that the banded pattern of unheated culture fluid was completely lost after heating, and only the labelled MPB70 preparation retained most of the radioactivity in a fraction with soluble protein of the same size. Most mycobacterial proteins are extensively denatured by these procedures, which explains the previous extensive difficulties in isolating defined constituents from PPD to characterize their behaviour in B- and T-cell reactions. In assays for the carrier effect of NIP-PPD for induction of anti-NIP production in BCG-vaccinated mice, the active fractions were heterogeneous in lectin reactivity as well as in SDS-PAGE. It appears most likely that a number of Mycobacterium tuberculosis proteins give rise to core peptides which resist proteolysis and heat denaturation to possess powerful T-cell-activating ability.

Specific immunosuppression by immunotoxins containing daunomycin.

Daunomycin, when conjugated with a targeting antigen by an acid-sensitive spacer, remains inactive at the intravascular pH of 7 but becomes active after cleavage within the acidic lysosomal environment of the target cell. This observation made it possible to construct cytocidal compounds that caused antigen-specific suppression of murine lymphocyte function. When daunomycin was coupled to the hapten conjugate of ovalbumin by an acid-sensitive cis-aconityl group, it caused hapten-specific impairment of immunocompetence in murine B lymphocytes in vitro and in vivo. Furthermore, the response by T lymphocytes to concanavalin A in vitro was selectively eliminated by a conjugate between daunomycin plus the acid-sensitive spacer and a monoclonal antibody specific for T cells.

Analysis of true anti-hapten cytotoxic clones in limit dilution microcultures after correction for "anti-self" activity: precursor frequencies, Ly-2 and Thy-1 phenotype, specificity, and statistical methods.

By the use of split culture techniques we have been able to demonstrate conclusively that the "anti-self" activity and the spontaneous anti-hapten activity within an apparent anti-hapten cytotoxic T cell response is a clonal phenomenon and is not caused by cross-reactivity between anti-self and true anti-hapten clones. With the knowledge of this clonality we have been able formally to prove that the true hapten-generated, hapten-specific response can be obtained by subtracting the response generated by "self" but directed against modified targets from the response generated by modified self and directed against modified targets. Unbiased statistical estimators, which do not make assumptions about the origin (0 responder cells, 100% negative cultures), have been developed to plot accurately limit dilution data (maximal likelihood estimator and minimal chi-square estimator), and the analysis demonstrates that all the components of an apparent anti-hapten response obey zero order (single hit) kinetics. By specifically identifying true anti-hapten and true anti-self clones, we have been able to study the phenotype of their precursors as well as the effectors themselves at the clonal level. Precursors of true anti-hapten and anti-self clones are Thy-1+, Ly-2+. However, anti-hapten and anti-self effector cells show marked clonal variation with respect to Ly-2, as some clones are almost completely insensitive to anti-Ly-2 and complement whereas others show minimal to complete sensitivity. All anti-hapten clones are completely sensitive to anti-Thy-1 and complement, whereas about one-third of anti-self clones show only partial sensitivity, with the most lytic clones showing the most sensitivity. Hapten-specificity and anti-self-specificity have been examined clonally. Ten percent of anti-TNP clones recognize NIP, 10% of anti-NIP clones recognize TNP, and 20% of anti-self clones recognize an allo-target. These figures are in accordance with the overall specificity of effectors generated in bulk culture in the presence of CAS (concanavalin A-stimulated spleen cell conditioned medium). However, hapten specificity and H-2 restriction of bulk generated effectors are improved if CAS is omitted from cultures.

Cell-mediated lympholysis of N-(3-nitro-4-hydroxy-5-iodophenylacetyl)-beta-anaylglycylglycyl-modified autologous lymphocytes. Effector cell specificity to modified cell surface components controlled by the H-2K and H-2D serological regions of the murine major histocompatibility complex.

Splenic lymphocytes from four C57BL/10 congenic mouse strains were sensitized in vitro to N(-3-nitro-4-hydroxy-5-iodophenylacetyl)-beta-alanylglycylglycyl-(N) modified autologous lymphocytes. The effector cells generated after 5 days of culture were assayed on a series of either N-modified phytohemagglutinin-stimulated spleen cells or N-modified tumor cells. The results indicated in all cases that both N modification of the targets and H-2 homology between the modified stimulating and target cells are required for lysis to occur. In each case the effector cells were found to lyse N-modified target cells only when there was homology at either or both ends of the major histocompatibility complex (MHC) between the stimulator and target cells. B10.BR lysed targets sharing alleles at K (or K plus I-A) and/or at D. B10.A effector cell specificity was mapped to K (or K plus I-A) and/or the D half of the MHC (D or D plus I-C and/or S). The two regions of specificity determined for B10.D2 effector cells were D (or D plus S plus I-C) and a region not including D of the MHC. C57BL/10 effector cells lysed N-modified targets only if there was target cell H-2 homology at K, I-A, and I-B or at the D serological region. As in the trinitrophenyl (TNP) system (6) B10.BR and B10.A effector cells lysed targets sharing K end H-2 serological regions greater than target cells sharing D-end serological regions. The C57BL/10 effector cells were shown to react to the K end greater than the D end, which differed from the equal reactivity seen in the TNP system for this strain. The data are consistent with the hypothesis that the antigen recognized by the effector cell includes an altered H-2 serological cell surface product. That the reaction is not "hapten specific" and the H-2 homology is required only for effector:target cell interaction was excluded by the use of two F1 combinations in which lysis of only N-modified target cells sharing the H-2 haplotype with the stimulating parental strain was obtained. Finally, it was demonstrated that N and TNP modification create distinct new antigenic determinants, since an effector cell sensitized to one modifying agent will lyse only H-2 matched target modified with that same modifying agent.

The role of B-cell memory in secondary IgG and IgM responses.

Mice were primed with the hapten 3-nitro-4-hydroxy-5 iodophenacetic acid (NIP) conjugated to chicken globulin (cg) and were boosted 2, 6, or 12 months later with CG conjugates of the related haptens 3,5-diiodo-4-hydroxyphenacetic acid (DIP) or 3-nitro-4-hydroxphenacetic acid (NP). Accelerated secondary responses were demonstrated both in the 7S and 19S class. Fine-specificities of secondary-response antibodies were studied by the hapten inhibition method of haptenated bacteriophage inactivation. 7S antibodies were found to have the fine-specificity of anti-NIP antibodies regardless of whether DIP or NP was the booster hapten ('original antigenic sin'). 19S antibodies had the fine-specificity of anti-DIP when DIP was the booster hapten. NP as the booster hapten resulted in 19S antibodies whose fine-specificity was intermediate between anti-NIP and anti-NP. A strong B-cell memory could thus be demonstrated in the 7S antibody response and a weak B-cell memory in the 19S antibody response.