Early thymic regeneration after irradiation.

Whole body irradiation produces profound thymic atrophy. After sublethal irradiation, regeneration begins promptly and the earliest regeneration is from radioresistant intrathymic precursors. The progeny of these precursors expand rapidly and restore thymic cellularity to near normal within 2 weeks. We have used monoclonal antibodies specific for a variety of differentiation markers of the T lineage to analyze the early events in thymic regeneration. A three-color flow microfluorometric analysis revealed that the majority of the cells found early in the regenerative process have the phenotype of mature T cells. These include CD4-/CD8-; CD3hi as well as CD4+/CD8-; CD3hi and Cd4-/CD8+; CD3hi. The proportion of cells with mature phenotypes declines rapidly between day 6 and day 12. Not all of the early appearing cells have mature phenotypes. Among the early cells that do not express CD3 are both CD4 and CD8 single positive cells that express HSA and resemble the intrathymic precursors found in other systems. In these mice CD4 single positive predominate. There are other cells that are HSA positive but express low levels of CD4 and very low levels of Thy-1. These appear to include the earliest members of the T-lineage. In addition to relatively mature conventional T cells and early progenitors, the early developing population includes cells that express markers of the T-cell lineage including the T-cell receptor but do not express Thy-1. These Thy-1 negative T cells comprise a significant number of the earliest cells found after regeneration.

Separation of self-renewing hematopoietic progenitors on the basis of CD45 (T-200) antigen expression.

Using monoclonal antibodies recognizing the shared determinants of the CD45 (T-200) antigen we have been able to distinguish spleen colony-forming units (CFU-s) whose progeny are self-renewing from other colony formers on the basis of their quantitative expression of this antigen(s). We have also been able to identify a population of cells that is capable of producing 12-day colonies but has only a limited capacity to produce 8-day colonies. CFU-s8 are found primarily in the dim T-200 population, whereas CFU-s12 were found in both the bright and dim population, but the cells within the colonies produced by these two populations differ in their capacity for self-renewal as CFU-s. Only the colonies dissected after 12 days from the spleens of mice receiving T-200-bright bone marrow cells contained significant numbers of cells that were capable of forming colonies after retransplantation. We calculate the frequency of these cells in total bone marrow to be approximately 1 in 5000.

Methods for the selection and growth of antigen-specific cytolytic T lines and clones bearing a defined T cell receptor beta chain marker.

Murine cytolytic T lymphocyte (CTL) clones specific for type A influenza virus antigens were generated by in vitro stimulation with syngeneic virus-infected cells in the presence of T cell growth factor (TCGF). All CTL clones recognize viral determinants shared by PR8 and X31 influenza viruses in association with a class I antigen, coded either by the H-2K or H-2D end of the appropriate haplotype. All clones express the Lyt2 antigen marker. Two of five clones also express an antigenic determinant of the V beta chain of the T cell receptor (TCR) identified by F23.1 monoclonal antibody. To effectively generate F23.1+ and antigen-specific CTL clones, heterogenous CTL lines were expanded with F23.1 coated Sepharose beads in the presence of TCGF and then stimulated with PR8 virus-infected cells. Thus, both the proliferative activity to PR8 and the expression of the F23.1 marker was increased significantly. Alternatively, F23.1+ T cells were sorted from in vivo primed mice and expanded with PR8 virus-infected stimulator cells in the presence of TCFG. This F23.1+ T cell line exhibited antigen-specific cytotoxicity for PR8 virus-infected target cells. Additionally, in an 'FcR-focused killing' assay only the F23.1+ CTL line and F23.1+ clones lysed Fc receptor bearing target cells in the presence of F23.1 antibody. These findings indicate that antigen-specific and F23.1+ clones can be selected with high efficiency by alternating stimulation with influenza virus-infected cells and with F23.1-coated Sepharose beads or through the use of a cytofluorograph. The usefulness of antigen-specific and F23.1+ CTL clones and other possible strategies for their selection are discussed.

Characterization of the fine specificity and antigen markers associated with the receptor of autoreactive T-cell hybridomas.

In this study we attempted to define the determinants on Ia molecules recognized by autoreactive hybridomas obtained from (C57BL/6 X BALB/c)F1 mice. The epitopes recognized by the T cells were characterized (a) using stimulating cells from various congenic and H-2 recombinant inbred strains and (b) by inhibition of activation with anti-Ia antibodies. Our hybridomas were strictly autoreactive and did not exhibit any alloreactivity, as is often observed for such cells. Our results show that more epitopes than previously believed are recognized by autoreactive T cells. One T-cell hybridoma (QW27.1) is unique in that it recognizes a hybrid F1 Ia determinant. Antigenic markers associated with the receptor of the T-cell hybridomas were studied with monoclonal antibodies (mAbs) specific for L3T4 and a V beta "idiotype". The results indicate that all Lyt 1.2 autoreactive T cells express L3T4 antigen in association with their receptor. One clone (QW64.14) expresses the V beta idiotype recognized by F23.1 monoclonal antibody. Moreover, this clone is activated by F23.1, linked to Sepharose 4B beads, which was believed previously to activate only Lyt 2+, L3T4 T cells. The supernatant of one clone (QW17.5) helps B cells to differentiate into antibody-producing cells without requiring direct contact with the autoreactive clone.

Suppression of the cytotoxic response of mouse lymphocytes to syngeneic tumor cells by tumor-promoting phorbol ester.

We have investigated the effects of tumor-promoting phorbol esters on the generation of T-killer cells against syngeneic tumor cells in tissue culture. C57BL/6 or BALB/c spleen cells were cultured with irradiated EL-4 or MPC-11 tumor cells, respectively, for 3 to 7 days at responder:stimulator ratios of 25:1 to 200:1. Lysis was measured in a 4-hr 51Cr release assay. During the sensitization phase, 12-O-tetradecanoylphorbol-13-acetate (TPA) at concentrations as low as 10 ng/ml inhibited the response by 90 to 100% at all responder: stimulator ratios and when added on Day 0, 1, 2, or 4 of a 5-day assay. Thus, TPA was able to suppress the response following successful activation of lymphocytes, since cytotoxicity could be detected as early as Day 3. Addition of TPA on Day 0 caused complete suppression of lysis when measured on Day 3, 5, or 7, indicating that the suppression was not due to a change in the kinetics of the cytotoxic response. The degree of suppression caused by five different phorbol compounds was positively correlated with their tumor-promoting activity. TPA was much less suppressive when added at the effector phase. Indomethacin, an inhibitor of prostaglandin synthesis, did not reverse the TPA effect even when added daily, beginning 3 days before the addition of TPA. The data suggest that one mechanism of phorbol ester tumor promotion may be the inhibition of T-cell immunity against tumor cells initiated by carcinogens.