Edgar E. Hanna

Deregulation of mouse antibody-forming cells by streptococcal pyrogenic exotoxin. II. Modification of spleen t-cell-complemented nude mouse pfc responses.

Abstract Streptococcal pyrogenic exotoxin (SPE), a toxic protein, secreted by Group A streptococci modifies antibody responses in two ways. It suppresses the early peak plaque-forming cell (PFC) and serum antibody responses to sheep erythrocytes (SE) and it engenders a late burst of PFC detected at 12–14 days. We have termed the late phase a deregulated response. This effect has been observed in rabbits and NIH (+/+ and +/nu) mice. NIH athymic nude (nu/nu) mice exhibit the early suppressed response but do not show the late phase. In reconstruction experiments to delineate the responsible target site of SPE we have conferred upon the nude or nude spleen cells in vitro , +/nu PFC responsiveness to SE by transfer of +/nu spleen cells in vivo or by supplementation with +/nu spleen cells in Marbrook cultures. When this is done, complementation of nude PFC responses and their ability to exhibit a deregulated response after SPE treatment is conferred coordinately. Pretreatment of donor cells with a B-cell inhibitory dose of X-ray or with a B-cell inhibitory dose of anti-Ig serum + C′ does not inhibit complementation of nude cells to +/nu responsiveness. Moreover, such donor suspensions when treated with SPE retain the ability to complement and to confer upon nude cells the ability to exhibit the late burst of PFC (a deregulated response). A similar pretreatment of the donor cell suspension with an anti-T-cell serum and C′, however, markedly inhibits both the adoptive complementation and the deregulation of the nude mouse PFC response. Thus, it is demonstrated that the target cell affected in this way by SPE is a T-cell. We presume from this evidence that SPE inhibits a T-cell which is involved in the regulation of antibody formation.

Suppression of the cytotoxic T-lymphocyte response in mice by pertussis toxin

Pertussis toxin (PT), the major toxin produced by Bordetella pertussis, has been reported both to enhance and to suppress immune responsiveness. These findings suggested that PT contributes to the virulence of B. pertussis through mechanisms involving immune regulation. We report that PT suppressed both the primary and the secondary cytotoxic T-lymphocyte (CTL) responses of mouse spleen cells cultured against two different allogeneic stimulator spleen cells in vitro. This suppression was dependent on the dose of PT used. PT must be present during the initial stages (within the first 24 hr) of CTL generation. Soluble factor(s) obtained from spleen cells preexposed to PT did not suppress the CTL response. Suppression of the CTL response observed was not due to depletion of the antigen by PT. The cytotoxic activity of CTL clones could not be suppressed by PT. The analysis of responder spleen cells, fractionated by anti-immunoglobulin panning techniques, provided evidence that L3T4-, Lyt 2+ cells mediate the PT-induced immunosuppression. We propose that suppression of the CTL response by PT is generated through the activation of L3T4-, Lyt 2+ suppressor T lymphocytes.

Proteolytic degradation of protein kinase C in the phorbol ester-induced interleukin-2 secreting thymoma cells

Effects of phorbol 12-myristate 13-acetate (PMA) on the fate of protein kinase C in two mouse thymoma cell lines, which are either responsive (EL4) or unresponsive (IEL4) to PMA-induced interleukin-2 (IL-2) production, were investigated with polyclonal antibodies raised against rat brain enzyme. These antibodies immunoprecipitated completely the protein kinase C from both cell lines and detected mainly an 82-kDa protein by immunoblot analysis of the crude homogenates as well as the partially purified kinase preparations. PMA elicited a time- and dose-dependent redistribution of protein kinase C from cytosol to the particulate fraction and proteolytic degradation of the kinase from both cell lines. The dose of PMA required for half-maximum protein kinase C translocation and degradation was at least five times lower for EL4 than for IEL4. In the presence of 16 nM PMA the rates of protein kinase C translocation and degradation were faster in EL4 than in IEL4, and the half-lives of protein kinase C in EL4 and IEL4 were less than 5 min and greater than 2 h, respectively. Analysis of the tryptic fragments of the immunoprecipitated enzyme, previously phosphorylated in the presence of [gamma-32P]ATP, revealed minor structural differences between the protein kinase C from these two cell lines. In neither cell line did the PMA-induced degradation of protein kinase C result in an accumulation of the Ca2+/phospholipid-independent kinase (catalytic unit) as analyzed by immunoblotting and gel filtration chromatography. Thus, activation of protein kinase C through the proteolytic conversion to the effector-independent catalytic unit plays little role in IL-2 production. The role of protein kinase C translocation and degradation in the PMA-induced responses in EL4 cells is unknown. However, IL-2 production in EL4 cells was reduced when PMA-induced degradation of protein kinase C was retarded by exogenously added protease inhibitors.

B-cell amplification by dibutyryl cyclic AMP in mice

Abstract Mice injected with N 6 -2′- O -dibutyryl-adenosine 3′,5′ monophosphate (DBcAMP) showed increased anti-sheep erythrocyte (anti-SE) antibody plaque-forming spleen cell (PFC) responses up to 7-fold greater than control mice. The amount of increase was related to the immunizing dose of SE and to the dose of DBcAMP, and it was more pronounced in 19S PFC than in 7S PFC. Mice thymectomized (Tx) within 16 hr after birth and injected with SE and DBcAMP at 40 days showed a 2.7-fold greater anti-SE PFC response than Tx controls injected with SE alone. DBcAMP restored the PFC response of Tx mice to 75% of that seen for sham Tx, DBcAMP-treated controls. These data suggest that a T cell-B cell interaction is not stringently required in mouse anti-SE antibody responses in vivo , since a T cell-like effect may be substituted or a minimal response can be enhanced with a soluble amplifier such as dibutyryl cyclic AMP.

Deregulation of mouse antibody-forming cells by streptococcal pyrogenic exotoxin (SPE): IV. Fractionation of a T-cell subpopulation which generates SPE-induced deregulation of anti-TNP PFC responses

Abstract Deregulation of the immune response to T-cell-dependent immunogens is a relevant biological property of Group A streptococcal pyrogenic exotoxin (SPE). The deregulated response induced by SPE differs from a normal regulated immune response to a T-dependent immunogen, trinitrophenylated rabbit erythrocytes (TNP-RE), by the generation of a late burst of plaque-forming cells (PFC) at 10–12 days without further immunogen stimulation. An in vitro cellular complementation system has allowed us to perform reconstruction experiments using the separate subpopulations of mouse immunocytes which include the B cells, T cells, and macrophages required for a complete anti-TNP PFC response in Marbrook-type cultures. Utilizing this system, the target of SPE activity was localized to the T-cell subpopulation. In this report we have fractionated two pools of spleen T lymphocytes from macrophage-depleted NFR/N +/nu mouse splenocytes via nylon wool columns. Analysis of these pools by cellular complementation, immunofluorescence, and antibody-complement-mediated inactivation showed the cell pools to contain different phenotypes. The subpopulation of T cells which eluted first (designated Pool 1) from the nylon wool column was capable of complementing the PFC responses of congenic NFR/N nu/nu mouse splenocytes. This result is consistent with their identity as helper T cells. The population of T cells which eluted second from the nylon wool column was designated Pool 2 and was capable of inhibiting the TNP-specific PFC responses of cultures optimally complemented with Pool 1 cells. This result is consistent with the identification of Pool 2 cells as a subpopulation with a phenotype as native negative regulatory T cells. Treatment of Pool 2 cells with SPE inhibited their suppressive activity for Pool 1 cells, and thus delineated the site of SPE-generated deregulation to Pool 2 cells.

Construction of hybridomas from nude mice with phenotypes of precursors of regulatory T cells: I. A model for study of development of T-cell function in immune systems

Functional helper and suppressor hybridomas were constructed from fractionated Type II NFR/nude mouse splenocytes which resemble precursors of T cells. After carrier priming with rabbit erythrocytes (RE), splenocytes from Type II NFR/nude mice were fractionated on nylon wool columns. Cells from two distinct fractions (Pool 1 and Pool 2) were fused with BW5147 lymphoma cells. Hybrids were selected in HAT medium and subsequently cloned. Three hybridomas constructed using Pool 1 splenocytes were capable of complementing nude mouse PFC responses to the trinitrophenyl group (TNP) of TNP-RE. Four hybridomas constructed using Pool 2 splenocytes were observed to suppress anti-TNP PFC responses. These hybridomas express the T-cell surface markers TLa, Thy 1, Lyt 1, Lyt 2 in patterns which suggest that they represent expanded clones of T cells from precursors that are arrested in their development. They appear to be blocked short of development of carrier specificity in their functional phenotypes as regulatory T cells.

Suppressor T-cell hybridomas that exhibit various capacities to negatively regulate PFC responses in a T-cell-dependent mouse model

Abstract Splenocytes which eluted second from nylon-wool columns (designated Pool 2) obtained from NFR/N mice that had been carrier primed with rabbit erythrocytes were fused with cells from the AKR T-cell lymphoma, BW5147. Several hybrid cell lines were obtained. Some of the cloned hybrids possess a marked capacity to negatively regulate (suppress) the T-cell-dependent hapten-specific plaque-forming cell responses of NFR/N nude mice that have been optimally complemented with littermate helper T cells or with a helper T-cell hybridoma cell line. The Pool 2 hybrids carry the Thy 1.2 marker of the immune parent. All of the Pool 2 hybrid lines tested to date express the Lyt2 surface marker. Interestingly, at least one of the cell lines expresses the Lyt1 surface marker concurrently. In addition, these hybridoma cells also express an Ig-V region determinant at their surfaces as detected by an anti-V H MOPC 315 antiserum as previously reported for cells of several Pool 1 T-cell hybridomas expressing helper phenotypes. Some of these hybridoma cell lines also express an Fc receptor for immunoglobulin.

Deregulation of mouse antibody-forming cells by streptococcal pyrogenic exotoxin (SPE): III. Modification of T-cell-dependent plaque-forming cell responses of mouse immunocytes is a common property of highly purified and of crude preparations of SPE

Abstract Group A streptococcal pyrogenic exotoxin (SPE) is a potent modulator of the immune system when used experimentally in mice. Typically, a late burst of plaque-forming cells (PFC) follows an early suppression of the antibody response in appropriately immunized and SPE-treated mice or their spleen cells in vitro. This altered response to antigen caused by SPE is termed a deregulated antibody response. The site of action of SPE was studied by use of cellular reconstruction and complementation experiments using the separated subpopulations of immunocytes which are required for full expression of mouse spleen PFC responses to sheep erythrocytes or to trinitrophenylated (TNP) rabbit erythrocytes in vitro. The SPE site was thus localized to the T-cell subpopulation. Recently SPE has been purified to a very high degree, making it possible to ascertain that SPE alone generates the deregulation of the immune system as described before and to limit the role of nondefined components of cruder preparations of SPE. A purified horse anti-scarlet fever antitoxin which recognizes highly purified SPE as being homogeneous also recognized a single component of crude SPE by agar-gel analysis. A rabbit anti-SPE immunoglobulin raised against crude SPE and absorbed with killed, strain NY5, Group A streptococci recognized the pure SPE and a major component of the homologous crude SPE similarly. Both of these antisera neutralized the capacity of SPE to deregulate the in vitro PFC response to TNP almost completely. A third antiserum raised in rabbits against a NY5 Group A streptococcal whole cell vaccine recognized a different component of crude SPE and totally failed to recognize pure SPE. This antiserum also recognized a purified Group A streptococcal peptidoglycan as being related to components contained in the crude SPE preparation. This antiserum, however, totally failed to neutralize the capacity of SPE to deregulate the PFC response to TNP. These results show that SPE-A is the active component of cruder preparations of SPE which deregulates PFC responses.

Complementation of the anti-TNP PFC response of N:NIH nude mice from nude dams by spleen cells of nude mice from heterozygous dams

Abstract Type I and Type II nude (nu/nu) mice are borne and raised by nu/nu dams and +/nu dams, respectively, under SPF conditions. Splenocytes from Type I nude mice exhibit even smaller in vitro PFC responses to trinitrophenylated rabbit erythrocytes than splenocytes from Type II nude mice. Type II splenocytes complement the magnitude of the PFC response of Type I splenocytes. The helper cell-like activity contained in Type II splenocyte suspensions was increased by treating Type II nude donor mice with dibutyryl cyclic AMP, but was essentially unaffected by pretreatment of Type II donors with endotoxin (LPS). The helper-like activity of Type II splenocytes was suppressed by treatment with a rabbit antiserum (plus C′) raised against a mixture of neonatal mouse thymocytes (RAM-T). We interpret the data to indicate that Type II splenocytes contain a larger number of residual T cells and/or a more fully developed population of T precursor cells that initiate or that are capable of being induced to initiate a helper function.

Origin of antibody-dependent, lymphoid-cell-independent hemolytic plaques in rabbit spleen cell cultures.

Summary This report describes a phenomenon that may be a significant source of error in the data acquired from hemolytic plaque assays of lymphocyte cultures. The data show that background antibody in the normal-serum supplement of the culture medium does not actually support initiation of a vigorous primary-type plaque-forming cell (PFC) response to sheep erythrocytes (SE). Instead, the background antibody causes the development of numerous spurious lymphoid-cell-independent plaques (LCIP). In general agreement with the literature, primary-type rabbit spleen cell responses in vitro were low in culture media supplemented with fetal bovine serum, a serum which does not contain background antibodies to SE. We thank Drs. P. Leder, M. Cashel, and S. G. Bradley for criticizing the manuscript and Ms. Catherine Kunkle for typing it.SummaryThis report describes a phenomenon that may be a significant source of error in the data acquired from hemolytic plaque assays of lymphocyte cultures. The data show that background antibody in the normal-serum supplement of the culture medium does not actually support initiation of a vigorous primary-type plaque-forming cell (PFC) response to sheep erythrocytes (SE). Instead, the background antibody causes the development of numerous spurious lymphoid-cell-independent plaques (LCIP). In general agreement with the literature, primary-type rabbit spleen cell responses in vitro were low in culture media supplemented with fetal bovine serum, a serum which does not contain background antibodies to SE.We thank Drs. P. Leder, M. Cashel, and S. G. Bradley for criticizing the manuscript and Ms. Catherine Kunkle for typing it.