40 immunology today, March 1981 ) Carrier-specific induction of hapten-specific suppression Leonore A. Herzenberg, Takeshi Tokuhisa and Leonard A. Herzenberg Department of Genetics, Stanford University School of Medicine, Stanford, California, 94305, USA Two functionally opposing, regulatory T cell popula- regulation of hapten-carrier responses. Therefore, our tions are found in carrier-primed mice. Both popula- data throw a substantially new perspective on the tions are raised by essentially the same immunization regulatory interactions involved in the response to protocol, both control antibody production to deter- common antigens. minants on typical carrier proteins, both are 'carrier- specific' in that they control responses to haptens The mechanism of carrier-specific suppression is added to the initial priming (carrier), and both are indeterminate commonly studied as regulators of such anti-hapten Carrier-specific suppressor populations were generally responses in adoptive or in-vitro assays. However, one presumed to regulate antibody production by control- population helps the antibody response while the ling the supply of carrier-specific help because they other substantially suppresses it. suppress in-vitro or adoptive in-vivo responses to 2, 4- These two populations have captured the limelight as regulators of heterogeneous antibody responses to T-dependent antigens. The helper T cells, because 150~ they are required for a successful response to these antigens, are generally thought to stimulate and thereby to directly control the expression of anti- hapten memory B cells. In contrast, the suppressor cells are thought to regulate B-cell expression indirectly by reducing the size or activity of the avail- I O0 able helper population. In addition, helper cells are DNP-KLH DNP-KLH thought to regulate suppressor activity through 1 O0 ug 1 ug homeostatic 'feedback loops' that can prevent exces- IgG2a on alum aqueous sive suppression. Thus, over the last few years, a anti complex but fairly consistent picture of response DNP regulation has evolved which places carrier-specific regulation at the center of the immunological uni- 5(2~ verse 1. Nevertheless, as we shall show, this picture is based on data which can (and probably should) be interpreted quite differently. Our recent studies 2 on KLH first the regulation of it~-situ responses to hapten-carrier conjugates reveal assumptions that mar previous con- clusions about the role of the carrier-specific -1 0 1 2 3 4 5 6 7 8 suppressor population and suggest a novel alternative Weeks for the contribution these cells make towards regulat- ing antibody production. Furthermore, the charac- Figure l. Carrier-priming prevents hapten-carrier stimula- tion of anti-hapten antibody responses. teristics of response regulation revealed by our studies (BALB/c x SJL) FI responses shown are representative of suggest that mechanisms akin to idiotype-specific and responses in BALB/c, BAB/14 and G3H. SW mice. Protocol and allotype-specific regulation may be central to "the assay details are described in the legend to Fig. 2. Elsevier / Non h-Ilolland Biomedical Press 1981 immunology today, March 1981 41 dinitrophenyl (DNP) on the priming carrier but are Anti-DNP responses are suppressed in ineffective in controlling antibody production by the KLH-primed mice exposed to DNP-KLH same B-cell population to DNP on an unrelated carrier. This interpretation of the suppression specifi- K ~ I 3 city data is consistent with the demonstration that allotype suppressor T cells remove allotype-specific K,DK -I f,o.31 i help and is reinforced by evidence that idiotype K,DK,DK 00"3) I suppressor T cells remove idiotype-specific help. These data have been taken as evidence that carrier- DK --i (5.) specific suppressor T cells interfere with the carrier- DK,DK l(too? i specific help interactions necessary for the production i I I I I I of antibody to determinants on the carrier molecule. 50 100 150 100 200 300 We suggest, however, that the logic underlying this Igh-la Anti DNP Igh-la Anti KLH analysis is flawed. The suppression of adoptive and in- (ug/ml) (units) vilro responses to DNP on the priming carrier, but not *K = KLH; DK = DNP-KLH on an unrelated carrier molecule, clearly demonstrates Fig. 2. Hapten-specific suppression selectively reduces the that the suppressor population is carrier-specific magnitude and affinity of anti-hapten responses. (since its function depends on being confronted with Animals were exposed to 100pg of the indicated antigens (on hapten on the appropriate carrier). But this specificity alum) at 6 week intervals. Responses were measured two weeks is consistent with a variety of mechanisms and does after the last indicated exposure. Units of ariti-carrier antibody are measured in radioimmunoassays (RIA) and calculated relative to not necessarily indicate that the suppressor cells a standard adoptive secondary response serum (to DNP-KLH). interfere with carrier-specific help for antibody Anti-DNP responses were measured in an RIA modified to responses to determinants on the carrier molecule. measure magnitude and affinitylL Data in parentheses after anti- Theoretically the suppressor population could DNP response 'bars' show the average K a M -~ x 106 for that establish a carrier-specific induction mechanism using response. the hapten-carrier conjugate to induce a second indicated, that carrier-primed animals produce a (hapten-specific) mechanism to specifically suppress minimal anti-hapten response when stimulated with the production of anti-hapten antibodies. This hapten on the priming carrier (Figs. 1 and 2). By the hapten-specific mechanism could operate ana- time we discovered the original papers which sug- logously to idiotype-suppression mechanisms by gested that the minimal responses were due to inter- specifically preventing the production of antibodies ference with anti-hapten memory development 5, we that have combining sites for the hapten and could had shown that anti-DNP memory development therefore suppress anti-hapten responses without occurs normally in keyhole limpet hemocyanin interfering with carrier-specific help or with the (KLH)-primed animals exposed to DNP-KLH (Table response to other determinants on the hapten-carrier 1) and that the anti-hapten responses fail because ex- conjugate. Consequently, carrier-specific help could posure to the hapten-carrier conjugate induces a be present and functioning normally in the presence of specific and persistent suppression of antibody p'ro- the suppressor population even though anti-hapten duction to the (new) DNP hapten on the carrier mole- antibody production is suppressed. cule (Fig. 3). This theory seems complex when compared with the relative simplicity of the hypothesis that carrier- The suppression-effector mechanism is hapten- specific help is depleted but the immune system, being specific more concerned with function than with appearance, The specificity of this suppression for responses to the may have a different idea of what constitutes a work- DNP hapten was established conclusively by the able set of regulatory interactions. In any event, as we observation that anti-DNP responses were suppressed shall show, a hapten-specific mechanism such as the in KLH-primed mice exposed first to DNP-KLH and one we have just described regulates antibody produc- then to DNP on an unrelated carrier molecule (i.e., to tion to hapten-carrier conjugates; and a carrier- DNP-CGG (chicken gamma globulin) ). These specific mechanism suppressing induction such as the animals produce normal primary responses to the one described operates in carrier-primed animals to CGG determinants on the DNP-CGG molecule and induce the hapten-specific system to suppress the thus have normal carrier-specific helper populations. response to DNP on the priming carrier. Furthermore, as indicated above, they have normal Our studies are rooted in a set of observations well anti-DNP memory populations. Therefore, if their known in the early 1970s but largely forgotten since anti-DNP responses were not specifically suppressed, then. We inadvertently repeated some in-situ studies they should have produced secondary level anti-DNP reported in the first papers on hapten-carrier help responses comparable with those in the control mice interactions 3,4 and found, as these papers had exposed to DNP-KLH and then to DNP-CGG. 42 immunology today, March 1981 TABLE I. Hapten-carrier stimulation of anti-hapten memory B minants on these antigens suggests that the hapten- cells is normal in carrier-primed mice~ specific mechanism normally operates to selectively control responses to each of the determinants on an immunizing molecule, regardless of whether the Memory B cell donors' form Adoptive IgG (haptenic)determinants are added chemically or are of antigenic stimulation anti-DNP response defined by the natural structure of the molecule. However, understanding this mechanism of action in Mean K a 'ordinary' immunizations (to haptens on the priming Group KLH DNP-KLH Units (x 10 6) carrier) first requires consideration of how it suppresses responses and makes animals persistently l * Alum 73 10 responsive or non-responsive to individual deter- II Alum Alum 73 8 minants on an antigen. III * Aqueous 18 0.7 The induction of hapten-specific suppression by the IV Alum Aqueous 50 8 KLH/DNP-KLH immunization sequence does not appear to be due to anything unique in the KLH carrier molecule or DNP hapten. Exposure to a GGG/DNP-CGG sequence induces similar suppres- Donors (BALB/c x SJL) were killed 3 weeks after the first indicated stimulation with DNP-KLH. Splenic B cells were prepared by sion for anti-DNP responses 6, and exposure to a eytotoxic depletionof T cells using monoclonalanti-Thy-1.2 (30-H12, KLH/PC-KLH sequence induces suppression of anti- J Ledbetter). Cells obtained from 107 spleen cells were co-transferred PC (phosphoryl choline) responses (J. Kenny, with 2 x 106 nylon-passedsyngeneic T cells from KLH-primed donors personal communication). Therefore, suppression (100 gg on alum plus 109 Bordetella pertussis at least 6 weeks before induction must represent the animal's response to ex- transfer).