The proliferative and anamnestic antibody response of rabbit lymphoid cells in vitro. I. Immunological memory in the lymph nodes draining and contralateral to the site of a primary antigen injection.

Popliteal lymph nodes were obtained from rabbits 4 days to 9 months after a primary injection of diphtheria toxoid or bovine gamma-globulin into the footpad. The ability of cells from these nodes to proliferate upon reexposure to antigen in vitro was compared to the height of the secondary response produced by tissue fragments. In addition, a comparison was made between the responsiveness of draining and contralateral lymph nodes. While the secondary antibody response in vitro increased markedly with the time after immunization at which the lymph nodes were taken from the animals, the degree of proliferation induced by antigen was highest with cells from lymph nodes taken early after priming (peak day 7) and was very much lower with lymph node cells taken longer than 3 wk after priming. This striking difference between these two responses has been discussed. Contralateral lymph nodes were much inferior to draining nodes in their ability to give a secondary antibody response in vitro, and never gave a detectable proliferative response. This difference became less marked with time after priming, but could still be demonstrated after 4 months. These results suggest a concentration of primed cells in the lymphoid tissue draining the site of injection, and a slow release of these cells into the circulation, to be distributed to the remaining lymphoid tissue.

Studies of PPLO infection. V. Inhibition of lymphocyte mitosis and antibody formation by mycoplasmal extracts.

Extracts of five arginine-utilizing mycoplasmas inhibit PHA-induced lymphocyte mitosis, while extracts of five glucose-utilizing mycoplasmas do not. Evidence is presented supporting the view that the inhibitory factor is the enzyme arginine deiminase. This enzyme inhibits the reactions of human lymphocytes to antigens as well as PHA, and the secondary production of antibody by rabbit lymph node fragments in vitro. Addition of enzyme to the cells several days after the initial mitotic or antigenic stimulus reduces, but does not abolish, further cellular activity. The production of serum proteins by hepatoma cells is totally unaffected by the mycoplasmal extract. It is concluded that arginine is an essential amino acid for the small lymphocyte, but not for the transformed cell nor for a number of other cell types. Suggestive evidence has been obtained that other enzymes similarly affect lymphocyte reactions.

Immunocompetent cells of the chicken. I. Specific surface antigenic markers on bursa and thymus cells.

Specific antisera to chicken thymus and to bursa of Fabricius were obtained in rabbits. After appropriate absorption and dilution all four anti-thymus sera, in the presence of guinea pig C', killed >90% of thymus and <==12% of bursa cells. They were cytotoxic for approximately 50% of spleen cells and did not affect antibody-forming cells. The surface antigen detected by these antisera was named chicken T-lymphocyte antigen (CTLA). Two of four anti-bursa sera, under similar conditions, killed >90% of bursa cells and <==10% of thymus cells. These antisera were cytotoxc for a large percentage of antibody-forming cells and killed approximately 30% of spleen cells The other two anti-bursa sera were somewhat less potent but showed similar specificity. The surface antigen detected by these antisera was named chicken bursa-derived lymphocyte antigen (CBuLA). Rabbit antisera to chicken immunoglobulin were cytotoxic for bursa but not for thymus cells and killed a similar percentage of spleen cells as did anti-bursa sera. They were also cytotoxic for antibody-forming cells.

Gamma globulin and antibody formation in vitro. VII. Effect of x-irradiation on the secondary antibody response in vitro.

The present studies have shown that the influence of X-irradiation on the secondary antibody response in vitro is remarkably similar to its effect on the primary response in vivo. When sensitized tissue was first irradiated and then reexposed to antigen, the duration of the interval between irradiation and antigen addition determined the degree of inhibition of the secondary response obtained. A delay of 12 hr resulted in stronger inhibition than a delay of 6 hr, and an interval of 24 hr before reexposure to antigen caused complete suppression of antibody production to diphtheria toxoid and almost complete suppression when sheep RBC were used as the antigen. Induction of the secondary response in rabbit lymph node tissue in vitro followed by exposure to X-irradiation, revealed that immediate exposure to irradiation after antigen produced stronger inhibition of the subsequent response than irradiation on days 2-3. Irradiation on day 6 had no detectable effect. The effectiveness of the early radiation is probably due to prevention of the proliferation of the antibody-forming cells. BUDR was found to be effective at similar time periods as X-irradiation, whereas colchicine could still stop antibody formation when added late during the secondary response in vitro. It was noted that lymph nodes from some BSA-sensitized rabbits as late as 18 months after sensitization gave a response indistinguishable from a typical secondary response, even when not reexposed to antigen.

Relationship of germinal centers in lymphoid tissue to immunological memory. I. Evidence for the formation of small lymphocytes upon transfer of primed splenic white pulp to syngeneic mice.

The fate, proliferation, and developmental potentialities of cell suspensions made from white pulp containing large germinal centers have been studied in the mouse by transfer of cells labeled with thymidine-(3)H to lethally irradiated, syngeneic recipients. Radioautographic analyses were made using both smears and sections of a variety of tissues. Thymidine-(3)H-labeling patterns of white pulp showed that, initially, labeling occurred in a majority of blast and "intermediate cells" but in very few or no small lymphocytes. After intravenous transfer, most of the labeled cells localized in the lymphoid tissues of spleen, lymph nodes, and Peyer's patches. Few cells migrated to the thymus, lung, liver, and intestinal mucosa. Both after intravenous and after intraperitoneal transfer there was a rapid increase in the incidence of labeled small lymphocytes and a decrease of labeled blasts and intermediate cells. This was accompanied by an increase in the grain count of the small lymphocytes and a progressive decrease in the grain counts of the blast cells. Exposure of nonlabeled donor cells to thymidine-(3)H at various time intervals after transfer indicated that dividing cells were present early after transfer but that their incidence progressively decreased. Between 24 and 48 hr, very little cell division was detectable.

Relationship of germinal centers in lymphoid tissue to immunological memory. II. The detection of primed cells and their proliferation upon cell transfer to lethally irradiated syngeneic mice.

White-pulp cells and whole spleen from donor mice immunized with sheep erythrocytes were transferred intravenously to heavily irradiated mice. The numbers of plaque-forming cells and the amount of hemagglutinating antibody produced after reexposure to antigen were measured. When reexposure to sheep erythrocytes was delayed, a much greater response occurred in the transferred cells. Peak responsiveness was reached at 24 hr after transfer. This "lag effect" was greatly reduced by repeated injections of 5-bromodeoxyuridine into the recipient mice prior to challenge with antigen. It was therefore concluded that much of the increase in responsiveness was due to a proliferation of "primed" cells after cell transfer. The fact that a significant response was given by the transferred cells in spite of 5-bromodeoxyuridine treatment suggested that some of the primed cells were nondividing. White pulp was a much richer source of responsive cells than was whole spleen.

The proliferative and anamnestic antibody response of rabbit lymphoid cells in vitro. II. Requirement for adherent and nonadherent cells of the responses to particulate antigens in spleen cell cultures.

Both primary and secondary responses to sheep erythrocytes and to Brucella abortus antigen have been obtained in cultures of dispersed rabbit spleen cells. Removal of adherent cells by repeated incubation of spleen cells on absorbent cotton diminished the ability of the spleen cell suspensions to give secondary as well as primary responses in vitro. When comparing cultures made in dishes and in tubes, the loss of responsiveness after incubation on cotton was much more evident in the dish cultures. It was concluded that the cell-to-cell interaction needed for immune responses to particulate antigens in vitro was more readily interfered with when the cells were spread over a larger surface area. The proliferative response to antigen, as measured by uptake of (3)H-thymidine in tube cultures of the sensitive spleen cells, appeared particularly resistant to the depletion effect of adherent cell removal. Dispersed spleen cells from sensitized mice gave a secondary response to sheep erythrocytes. This response was readily abolished by one incubation on absorbent cotton when the cells were cultured in dishes.

Surface antigens of immunocompetent cells. 3. In vitro studies of the role of B and T cells in immunological memory.

The effect of preincubation with anti-theta or anti-mouse immunoglobulin (Ig) and complement (C') on immune responsiveness of spleen cells from BALB/c mice immunized with sheep erythrocytes (SE) was investigated. Both treatments greatly depressed the remaining ability to produce a secondary response to SE in vitro. Normal BALB/c spleen cells were far less effective in reconstituting the responses of such depleted cell populations than were much smaller numbers of untreated immune spleen cells. Thymus-derived cell (T cell) memory appeared early after immunization and showed specificity for the immunizing antigens. Recombination of anti-Ig-treated with anti-theta-treated immune spleen cells resulted in virtually complete reconstitution of responsiveness. The presence of immunological memory in T cells and the nature of their surface receptors are discussed.

Thymus-derived cell (T cell) activation by heterologous antigens as a replacement of specific immune T cells in the transfer of the secondary response to sheep erythrocytes.

Spleen cells from LAF(1) mice hyperimmune to sheep erythrocytes (SE) lost their ability to transfer a secondary response to irradiated recipients after incubation with anti-theta and rabbit complement in vitro. Small numbers of specific immune cells even when taken 3 days after a primary SE injection reconstituted the direct and indirect plaque-forming cell responses. Larger numbers of cells sensitized to B. abortus (or keyhole limpet hemocyanin), and given together with the corresponding antigen, also partially reconstituted the ability to respond to SE. This property was mediated by theta-bearing cells and was interpreted as due to a nonspecific humoral factor liberated by specifically activated T cells and acting on B cell proliferation or maturation.

Surface antigens of immunocompetent cells. I. Effect of theta and PC.1 alloantisera on the ability of spleen cells to transfer immune responses.

Spleen cell transfer studies were done in BALB/c strain mice in an attempt to define the role of theta-antigen-bearing lymphoid cells in immune responses to SE. Incubation with alloantiserum to theta-C3H and rabbit C' virtually completely abolished the ability of the cells to transfer both primary and secondary (IgM and IgG) responses to 650 R irradiated recipients. Normal thymus cells partially reconstituted the ability of such treated cells to transfer the primary but not the secondary response. The results are interpreted as showing immunological memory for SE in the theta-bearing thymus-derived cells. Incubation of the spleen cells with alloantiserum to the PC.1 antigen present on antibody-forming cells did not significantly affect the ability to transfer either primary or secondary response.

The liver as the site of C-reactive protein formation.

The site of formation of C-reactive protein (CxRP, CRP) has been studied with tissues from rabbits, monkeys, and human beings. Rabbits and monkeys were stimulated to produce the acute phase protein by injection of turpentine, croton oil, endotoxin, paratyphoid-typhoid vaccine, or pneumococci. C(14)-amino acid incorporation in vitro was demonstrated by means of autoradiography of immunoelectrophoretic patterns made with culture fluids. It was found that among many different tissues tested liver was the only tissue which incorporated C(14)-lysine and isoleucine into CxRP or CRP. Only livers taken 16 to 24 hr after various stimuli were active; livers from normal animals or from animals killed 3 to 9 hr after stimulation did not produce detectable amounts of CxRP. Inflamed muscle from the injection site did not show C(14)-amino acid incorporation into CxRP. Several human tissues were also cultured, and a few liver cultures found to contain labeled CRP. The formation of CxRP or CRP by the liver was always accompanied by enhanced C(14)-amino acid incorporation into other serum proteins, but the reverse was not always found.

Transfer of antibody production with cells from bursa of Fabricius.

F(1) hybrid chicks isogenic for the strong B histocompatibility locus and for most weak H-loci were X-irradiated on day 1 after hatching, injected intraperitoneally on day 2 with dispersed cells of bursa, spleen, or thymus from 4- or 10-wk-old F(1) hybrid donors, and immediately challenged by the same route with either Brucella abortus, sheep erythrocytes, or a mixture of both together. The agglutinin titers were measured in sera obtained 1 wk later. With 4-wk-old donors, a greater primary response to Brucella abortus was obtained after transfers of cells from bursa than from spleen, while thymus was much less effective. With 10-wk-old donors, the decreasing order of response was spleen, bursa, thymus. Only splenic cells were effective in transferring a response to sheep erythrocytes, at either donor age. In tests of synergism by cell mixtures from pairs of organs, the only positive finding was a modest augmentation of titer against sheep erythrocytes by bursa + spleen as compared with spleen alone. Bursal cells from 6- or 10-wk-old donors were effective in transferring a response to sheep erythrocytes when antigen injection was delayed until 5 days after cell transfer. Splenic cells from hormonally bursectomized donors were ineffective in transferring a primary response, even when the donors had been injected with antigen 1 wk before transfer. Preimmunization of normal donors led to marked increases in the responses to Brucella abortus produced by transferred splenic or thymic cells. With bursal cells, an increased response was obtained only if the interval between preimmunization and transfer was 17 rather than 7 days. With the 17-day interval, both bursal and thymic cells could also transfer a response to sheep erythrocytes. The primary sera to Brucella abortus produced after transfers of bursal or splenic cells contained almost entirely 19S antibodies. A 7S component was found in all the secondary sera tested.

Selective viral immunosuppression of the graft-versus-host reaction.

Graft-vs.-host (GVH) reactivity of parental lymph node (LN) cells was assayed by measurements of 3H-thymidine incorporation in vivo in spleens of irradiated F1 recipients. Preincubation of parental LN cells with vesicular stomatitis virus (VSV) for 2 h at 37 degrees C followed by washing resulted in an 85-90% reduction in splenic radioactivity, as did injection of VSV on days 0-2 after recipients received untreated parental LN cells. In contrast, 3H-thymidine incorporation in the spleens or irradiated F1 hosts was not affected by VSV when F1 bone marrow cells were incubated with the virus. In addition, preincubation of F1 B cells with VSV still allowed these syngeneic B cells to be recruited into proliferation by mitomycin-treated parental LN cells. The inhibitory effect of VSV, thus, seems to be specific for T-cell proliferation. These observations suggest that viral immunosuppression might be capable of being developed into a useful strategy for selective deletion of lymphocytes capable of reacting against histocompatibility antigens and initiating GVH reactions.

Growth of SJL/J-derived transplantable reticulum cell sarcoma as related to its ability to induce T-cell proliferation in the host. I. Dominant negative genetic influences of other parent haplotype in F1 hybrids of SJL/J mice.

Growth of three transplantable reticulum cell sarcomas (RCS) was studied in a variety of F1 hybrids of SJL/J mice by determination of lymph node (LN) and spleen: body weights ratios 7 and 14 d after intravenous injection of RCS cells. Comparison of BIO.S x SJL and A.SW x SJL with SJL/J showed a negative effect of both the A and the BIO non-H-2 genes, particularly on growth in LN. F1 hybrid resistance was noted with F1 hybrids that carried H-2Dd and was much more evident with F1 hybrids from BIO- than from A-background mice. This resistance was less marked at 14 than at 7 d and was partially overcome by injection of higher tumor doses. Changing the I region in the F1 parent from H-2d to H-2b or H-2f had no effect on growth, but changing to H-2k or H-2d virtually abolished the ability to support tumor growth. This effect appeared partially as a result of the I-E/C and partially of the I-A(B) region and was not overcome by higher tumor dose or longer intervals after injection. There also appeared to be a negative influence on growth of H-2Kk, but this was difficult to differentiate from the I-Ak effect with the available strains. The known proliferative responsiveness that SJL/J Lyt-1 T cells exhibit to Ia determinants on gamma-irradiated RCS cells in vitro was also compared with that of cells from various F1 hybrids. Responsiveness of F1 LN cells was expressed as a percentage of the response in SJL/J LN cells to the same RCS cells, measured as [3H]thymidine incorporation. There was a striking degree of correlation between proliferative responsiveness of F1 LN cells to RCS and the ability of the F1 mice to support tumor growth. This correlation was especially clear with respect to the negative influences of non-H-2 genes, and of H-2 loci in the I region, particularly of I-Ak or -d and of I-E/Ck or -d, but there also appeared to be a (smaller) negative effect of I-Ab or -f. Negative influence of H-2Dd on growth, however, was not reflected in a similarly large effect on the proliferative response. Additional findings showed that LN cells from all F1 hybrids exhibited equivalent syngeneic mixed lymphocyte responses in the presence of polyethylene glycol to mitomycin-treated spleen cells from both the SJL/J and the other parent. The extra high response of F1 cells to RCS cells, as compared with SJL spleen cells, however, was always absent when Ik or -d was contributed by one of the F1 parents. The results suggest a promoting effect of the proliferative response on RCS growth in vivo and, furthermore, an interesting effect of I-A and I-E/C genes, possibly via an interaction product, on the ability of LN cells to be stimulated by Ia determinants on RCS cells.

Physiology of IgD. VI. Transfer of the immunoaugmenting effect of IgD with T delta-containing helper cell populations.

We show that the IgD-induced augmentation of the immune response to trinitrophenylated keyhole limpet hemocyanin can be transferred to syngeneic mice with spleen cells from IgD-injected donors. The augmenting activity is present in the Lyt-1+2-, L3T4+ T cell population and is absent from B cells. The ability of transferred T cells to augment the immune response correlates with the presence of a high frequency of Lyt-1+2- T cells that form rosettes with IgD-coated sheep erythrocytes (T delta cells). Such rosette-forming cells can also be induced by incubation of spleen cells from normal donors in IgD-coated petri dishes. Injection of normal spleen cells exposed to IgD-coated petri dishes together with antigen also augments the immune response of recipients. The existence of a regulatory circuit based upon interactions between T delta cells, antigen, B cell surface IgD, and serum IgD, is proposed.

Production of auto-anti-idiotypic antibody during the normal immune response to TNP-ficoll. III. Absence in nu/nu mice: evidence for T-cell dependence of the anti-idiotypic-antibody response.

Although athymic mice make an excellent immune response to the thymus-independent antigen trinitrophenyl-lys-Ficoll (TNP-F), nude mice of AKR/J and BALB/c strains lack the anti-idiotypic response that occurs in euthymic mice of both of these strains within the first 1--2 wk after injection of more TNP-F. Anti-idiotypic antibody-blocked (hapten-augmentable) anti-TNP splenic plaque-forming cells (PFC) do not occur at any time and serum anti-idiotypic antibody is absent in both congenitally athymic mice, and thymectomized, irradiated, bone marrow-reconstituted mice. Nevertheless, nu/nu mice do have PFC which can be inhibited by exposure to anti-idiotypic antibody produced in +/+ mice. As a consequence of the failure to produce anti-idiotypic antibodies, the anti-TNP PFC response is athymic as compared to euthymic mice is of greater magnitude, declines less precipitously, and shows an increase rather than a decrease in affinity between days 4 and 7 after antigen injection. It is concluded that the anti-idiotypic antibody response is thymus dependent and that athymic mice lack a helper cell required for the induction of anti-idiotypic antibodies.

Production of auto-anti-idiotypic antibody during the normal immune response to TNP-Ficoll. II. Hapten-reversible inhibition of anti-TNP plaque-forming cells by immune serum as an assay for auto-anti-idiotypic antibody.

Sera taken from AKR/J mice 7 d after the intravenous injection of 2,4,6-trinitrophenyl-lys-Ficoll (TNP-F) caused a specific inhibition of anti- trinitrophenol (TNP) plaque-forming cells (PFC) in vitro. This inhibition was reversed by the incorporation of 10(-8)-10(-7) M 2,4,6-trinitrophenyl- epsilon-amino-n-caproic acid (TNP-EACA) into the agar during the PFC assay. The factor responsible for the hapten-reversible PFC inhibition was removed from serum by passage through an anti-immunoglobulin column or through a 2,4,-dinitrophenyl-human-serum-albumin-bromoacetylcellulose plus anti-TNP- antibody column, but not by DNP-HSA-BAC alone. It was concluded that this immunoglobulin-like substance, lacking anti-TNP activity but reacting with anti-TNP antibody of AKR/J origin, was most likely an auto-anti-idiotypie antibody that had been produced during the normal course of the response of AKR/J mice to TNP-F. Pools of anti-idiotypic-antibody-containing antisera inhibited anti-TNP plaque formation to varying degrees when tested on d-4 PFC from different mice of the same inbred strain, suggesting a variability in idiotype expression. 4 d after transfer of immune (7 d after 10 mug TNP-F, administered intravenously) AKR/J spleen cells plus 10 mug TNP-F into syngeneic mice, the number of PFC detectable in the recipients' spleens could be markedly augmented by the inclusion of TNP-EACA in the agar during the PFC assay. Incubation of spleen cells containing such hapten-augmentable PFC with TNP- EACA yielded a factor in the supernate that caused a specific, in vitro, hapten-reversible inhibition of anti-TNP PFC. Studies with immunoadsorbents indicated that this PFC-inhibiting factor was antigenically immunoglobulin- like, lacked anti-TNP-antibody activity, but reacted with anti-TNP antibody of AKR/J origin. The results are consistent with the view that this PFC inhibitor is auto-anti-idiotypic antibody that is involved in the normal regulation of the immune response. It is proposed that hapten-reversible inhibition of plaque formation can be employed as an assay for anti-idiotypic antibody and the conditions for such an assay are described. It is further proposed that the detection of hapten-augmentable PFC suggests the presence of auto-anti-idiotypic antibody.

Production of auto-anti-idiotypic antibody during the normal immune response to TNP-ficoll. I. Occurrence in AKR/J and BALB/c mice of hapten-augmentable, anti-TNP plaque-forming cells and their accelerated appearance in recipients of immune spleen cells.

Attempts were made to elucidate the cause of the downward regulation of the splenic plaque-forming cell (PFC) response in AKR/J and BALB/c mice between days 4 and 7 after a single intravenous injection of 2,4,6,trinitrophenyl- lys-Ficoll(TNP-F). AKR/J spleen cells, taken 7 d after injection of TNP-F, were transferred, together with TNP-F, into normal AKR/J mice. The day-3 or - 4 PFC response of the recipients was much lower than that of recipients of normal cells. However, the suppression was only apparent because the presence of 10(-8)-10(-7) M 2,4,6-trinitrophenyl-epsilon-amino-n-caproic acid (TNP- EACA) (or 10(-7)-10(-6) M 2,4,-dinitrophenyl-epsilon-amino-n-caproic acid) in the PFC assay caused a dramatic increase in observed PFC, averaging 298 percent on day 3 and 122 percent on day 4. Recipients of normal cells showed no such hapten-augmentable PFC. T-depleted immune spleen cells did not cause any apparent suppression of the response to TNP-F, but hapten-augmentable PFC in recipient spleens were again prevalent. Suppression of the PFC response, as well as hapten-augmentable PFC, were seen after transfer of immune serum. It was postulated that hapten augmentation of PFC was caused by displacement of auto-anti-idiotypic antibody from the surface of blocked antibody- synthesizing cells. Further studies showed that such hapten-augmentable PFC occurred in the spleens of a large percentage of both AKR/J and BALB/c mice examined after day 4 of the primary response to TNP-F. Thus, it was hypothesized that the downward regulation of the magnitude and, possibly, also of the heterogeneity of the splenic-PFC response was due to an auto-antibody response to one or more major idiotypes of the anti-TNP response.

Properties of reticulum cell sarcomas in SJL/J mice. V. Nature of reticulum cell sarcoma surface antigen which induces proliferation of normal SJL/J T cells.

The results of studies on the reticulum cell sarcoma (RCS) tumors of SJL/J mice presented here, indicate that spontaneous tumors, which arise in older mice, also possess the capacity to induce the vigorous proliferative response in syngenetic T lymphocytes that are characteristic of the transplantable RCS lines. Analysis of cell surface antigens revealed the presence of Ia determinats on gradient-purified transplantable RCS tumor cells; however, these cells did not express Thy 1.2, nIg, or, any of the viral proteins that were tested for by specific antisera. Pretreatment of RCS cells with anti-Ia sera and complement-deleted cells that were stimulatory for syngenetic T lymphocytes, and addition of anti-Ia sera directly to cultures blocked the proliferative response at the stimulator (RCS) cell level. Lymph node cells from H-2(8) strains other than SJL/J, including A.SW and B10.S also gave proliferative responses to RCS cells, although lower in magnitude. A requirement on the part of responding cells for identity with RCS cells at the Ir region was indicated by the finding that A.TH but not A.TL lymph node cells responded to RCS. It is concluded that RCS cells stimulate Ir-region identical T cells (without evidence of presensitization) through a modification in the expression of Ia antigens on the surface of the tumor cells.

Physiology of IgD. IV. Enhancement of antibody production in mice bearing IgD-secreting plasmacytomas.

Immune responses to trinitrophenylated hemocyanin (TNP-KLH), Ficoll (TNP-Ficoll), and Brucella abortus (TNP-BA) were examined in BALB/c mice bearing subcutaneous transplants of TEPC-1017 and TEPC-1033, the two known IgD-secreting BALB/c plasmacytomas. Both primary and secondary 19S and 7S splenic plaque-forming cell (PFC) responses in spleen to intravenously injected TNP-KLH were enhanced three to fivefold. Primary responses to TNP-Ficoll were 1.5-2 times higher than in control mice (particularly the 7S PFC response). Primary responses to TNP-BA were enhanced by TEPC-1017 but suppressed by TEPC-1033, while secondary responses to TNP-BA were enhanced three to sevenfold by both tumors. Intraperitoneal injections of ascites fluid from mice bearing TEPC-1017 or TEPC-1033, or of IgD isolated from such ascites fluid, caused a similar enhancement of the primary response to TNP-KLH, as did the tumor itself, particularly when injected approximately 1 wk before antigen injection. IgD-containing ascites fluid had no effect on the response of athymic (nu/nu) BALB/c mice to TNP-KLH. These findings suggest the existence of an IgD-responsive immunoregulatory T cell.