Adoptive transfer of allotype-specific suppressor cells inhibits thymus-independent immunoglobulin production in syngeneic athymic mice.

(SJL X BALB)F1 suppressed mice have, in their lymphoid tissues, a population of suppressor T cells directed specifically against a paternal gamma G2a allotype (Ig-1b). Spleen or lymph node cells from these mice were injected into syngeneic nude mice and the effect on thymus-independent synthesis of Ig-1b in the athymic recipients was determined. After the injection of suppressor cells, Ig-1b disappeared from the serum of the recipients in a time course similar to that seen in normal mice. These results indicate that suppression occurs in the absence of thymus-derived helper cells, and they suggest that Ig-1b-producing B cells are the target of allotype-suppressor cells.

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.

Active suppression of immunoglobulin allotype synthesis. I. Chronic suppression after perinatal exposure to maternal antibody to paternal allotype in (SJL x BALB-c)F 1 mice.

Long-term (chronic) allotype suppression, previously reported only in rabbits, is shown here to occur in at least one strain combination of mice as well. Close to 50% of the offspring of SJL (Ig(b)) males mated to BALB/c (Ig(a)) females immunized against the paternal allotype were found to be suppressed for Ig-1b (gammaG(2a)) at 6 months of age. These mice are called "chronically" suppressed. The percentage of offspring in this strain combination suppressed for the paternal allotype at 8 wk of age is the same as that seen in an earlier strain combination tested, [(C57 x BALB/c)F(1)], in which all mice recover from suppression by 10-12 wk. After 8 wk, two distinct patterns of long-term (chronic) suppression emerge in (SJL x BALB/c)F(1) mice: a small number of these mice never produce detectable amounts of Ig-1b throughout their lives, while the majority produce detectable Ig-1b sporadically, sometimes over a period of several weeks, the level of which eventually falls below detectability. Attempts to "cure" suppression by destroying the existent lymphoid population and forcing endogenous repopulation in chronically suppressed animals were unsuccessful. Furthermore, attempts to restore Ig-1b production by injection of cells from syngeneic Ig(a)/Ig(b) donors into irradiated, chronically suppressed recipients were also unsuccessful, although the same cell inocula, when injected into irradiated BALB/c (Ig(a)/Ig(a)) mice produced high levels of gamma globulin carrying the allotype. These results suggest that long-term allotype suppression resulting from perinatal exposure of offspring to specific anti-allotype antibody (anti-Ig-1b), is not due merely to an absence of Ig-1b-producing cells or their progenitors, but appears to be an active process, which dominates physiologically over normal production.

Immunological memory in mice. II. Cell interactions in the secondary immune response studies by means of immunoglobulin allotype markers.

Congenic mice, differing genetically only at the loci coding for immunoglobulin H chain (or Fc) structures, have been used to study cell interactions in the 7S (gammaG(2a)) antibody response to sheep erythrocytes (SRBC), as detected by the Jerne plaque-forming cell (PFC) method. The interaction between thymus and bone marrow cells was studied in adult thymectomized irradiated recipients, protected with syngeneic bone marrow and injected with thymus cells from the partner congenic strain. All of the gammaG(2a) PFC detected in the spleens of these mice were of bone marrow allotype. Adoptive secondary immune responses were then studied to determine whether a similar interaction between memory cells and bone marrow derived cells could be detected. Primed spleen cells from the partner congenic strain, or a subpopulation of these cells obtained by BSA density gradient fractionation, were injected into irradiated recipients alone, or together with syngeneic nonimmune spleen or bone marrow cells. All gammaG(2a) PFC detected in these experiments were of primed cell allotype. There was no evidence that antibody forming cell precursors in normal spleen or bone marrow participate in the adoptive secondary immune response detected 7 days after transfer of primed spleen cells. This was true regardless of whether the bone marrow cells were injected at the time of transfer, or were injected 1-2 wk earlier and allowed to become established in the spleens of recipient mice. Although no specific cell interaction was seen, bone marrow (and, to a lesser degree, normal spleen) cells were found to have a nonspecific enhancing effect on the adoptive secondary response when they were injected together with the primed spleen cells. This enhancement was not evident if the bone marrow cells were injected 1 or 2 wk prior to primed cell transfer.

Active suppression of immunoglobulin allotype synthesis. II. Transfer of suppressing factor with spleen cells.

The mechanism of chronic allotype suppression in (SJL x BALB/c)F(1) mice has been investigated by means of cell transfer studies. These mice are phenotypically negative for serum Ig-1b, the paternal allotype determinant on gammaG(2a) immunoglubulin, as a result of perinatal exposure to maternal anti-Ig-1b. When spleen or bone marrow (B) cells from suppressed mice were injected into irradiated BALB/c "indicator" hosts, detectable levels of Ig-1b were demonstrated in the sera of a majority of the recipients early after transfer. These results indicate that Ig-1b-producing cells or their precursors are present in the lymphoid tissues of suppressed mice, even though they are not expressed. Within 5-7 wk, it was no longer possible to detect Ig-1b in the sera of these hosts, although cells producing another paternal allotype (Ig-4b) were shown to persist. Control BALB/c mice, injected with spleen and B cells from normal mice, continued to produce high levels of immunoglobulin carrying this allotype. The disappearance of serum, Ig-1b occurred most frequently in the recipients of suppressed spleen cells. Similar results were obtained using a mixture of spleen cells from normal and suppressed mice. Ig-1b production in the recipient mice ceased within a few weeks, even though the majority of cells in the mixture were obtained from normal (nonsuppressed) donors. The data are interpreted as evidence that chronic allotype suppression in mice is actively maintained by cells which are resident in the lymphoid tissues, splenic cells being the most effective. These cells are capable of proliferating in a new host and exerting their suppressive influence on Ig-1b-producing cells and/or their precursors.

Physiology of IgD. I. Compensatory phenomena in B lymphocyte activation in mice treated with anti-IgD antibodies.

The role of delta-positive cells in the immune response was studied by comparing the effects of treatment with allotype-specific IgD hybridoma antibody on homozygous BALB/c or SJL/J and heterozygous (BALB x SJL)F1 mice. Homozygous mice, injected from birth with the relevant anti-delta antibody, made primary or secondary immune responses to intravenously injected trinitrophenyl (TNP)-Brucella abortus, TNP-Ficoll, and TNP-keyhole limpet hemocyanin, which did not differ significantly from those of control mice, despite the fact that IgD+ cells were depleted and Ig+ cells were markedly reduced in the spleens of treated mice. Responses in nodes draining a local injection of TNP-Brucella abortus were, however, significantly suppressed. Heterozygous mice, injected from birth with either anti-Ig-5a or anti-Ig-5b, showed a marked reduction in the number cells producing IgG antibody of linked allotype specificity in the secondary response to intravenously injected sheep erythrocytes. A corresponding decrease in the amount of serum IgG2a of that allotype specificity was also noted. However, in agreement with the results obtained in homozygotes, heterozygotes injected simultaneously with anti-IgD directed against each of the allotypes made normal, if not enhanced, plaque-forming cell responses of both allotype specificities. Similarly, serum IgG2a levels were normal in all but one mouse treated in this fashion. These results indicate that IgD+ cells are not essential for an immune response in vivo. Although the delta-positive cell is used preferentially under normal conditions, it appears that an alternative mechanism exists by which, in the absence of these cells, the animal is able to make a normal immune response.

Ontogeny of B lymphocyte function. XV. A role for thymus cells in the maturation of the capacity of a subpopulation of B cells to re-express surface immunoglobulin after treatment with anti-immunoglobulin.

The maturation of the C57BL/6 B cell population to be able to re-express surface immunoglobulin (sIg) after its removal by treatment with rabbit antimouse Ig (RAMIg) was studied in a cell transfer system. It was found that thymus cells were required for the maturation of a subset of the B cell population to be able to re-express sIg. The B cell population of irradiated, thymectomized mice reconstituted with spleen cells from donors under 2 wk of age remained deficient in their ability to re-express sIg even after 4 wk residence in the cell transfer recipient. In contrast, if adult thymus cells were transferred together with the immature B cells, the B cell population matured to be able to re-express sIg after treatment with RAMIg. Approximately one-third of the B cell population appears to require thymus cells for this maturation. The maturation of the thymus cell population to be capable of mediating this maturation of the B cell population occurs in two steps: between 2 and 3 and between 3 and 4 wk of age. This timing corresponds to the age at which the B cell population of C57BL/6 mice normally acquires the capacity to re-express sIg, which we have previously shown to also occur in two steps. Thymus cells from 3-wk-old donors can mediate the first step in B cell maturation to be able to re-express sIg, but cannot mediate the second step in this maturation of the B cell population. Thymus cells from 4-wk-old donors can mediate both steps in the maturation of the B cell population. The results suggest that thymus cells are involved in regulating some aspects of B cell differentiation.

Migration of peripheral T and B cells into the thymus of aging (NZB X SJL)F1 female mice.

Aging NZB X SJL (NS) female mice provide a unique model of thymus pathology characterized by the intrathymic accumulation of large numbers of mature T and B cells. The purpose of the present work was to examine the possibility that this phenomenon results from the invasion of the thymus by cells from the periphery. Lymphoid cells labeled with chromium-51 or indium-111 were injected into syngeneic recipients to study their patterns of in vivo migration. Lymph node (LN) or spleen cells were found to localize significantly (1-2% of injected radioactivity) into the thymus of 12-month-old NS females but not into that of young recipients or of old NS males. However, intrathymic localization of injected LN cells was observed in castrated NS males which exhibit the same thymus pathology as NS females. Both radiolabeled T and B cells were found to enter the thymus of aged NS females but the latter cells about three times less efficiently than the former. Moreover, while thymocytes from young NS females were unable to recirculate to LN, those of old NS females showed increased LN-seeking capacity and part (1%) of them did migrate back into the thymus of old but not young NS females. In additional cell transfer experiments, the intrathymic migration of B cells into old NS females was further documented by using the antibody response to sheep erythrocytes as a tracer. Taken together, these observations indicate that the thymus of aging NS female mice is permeable to recirculating lymphocytes, suggesting that at least part of the mature T and B cells detected in this thymus are migrants from the periphery.

Chicken T lymphocyte clones with specificity for Eimeria tenella. I. Generation and functional characterization.

T lymphocyte clones reacting specifically with the antigenic components of Eimeria tenella were generated from splenic lymphocytes of immunized chickens and were maintained for 12 to 14 wk in vitro. These T cell growth factor-dependent T lymphocyte clones from bursectomized and normal chickens proliferated in vitro when stimulated with antigens from different developmental stages of homologous but not heterologous species of the parasite. Specific proliferative responses of the cloned T cells showed an absolute requirement for antigen presentation by histocompatible antigen-presenting cells. Some of the T cell clones exhibited functionally discrete interactions with syngeneic primed B cells; 25% of the T cell clones from immunized normal chickens and 7% of those obtained from immunized bursectomized chickens showed antigen-dependent helper activity and induced specific antibody production by syngeneic primed B cells. Of the T cell clones from immunized normal chickens, 19% showed suppression of in vitro antibody production in comparison to 7% of those isolated from immunized bursectomized chickens. The frequency of cloned T cells with ability to induce cytotoxic activity in macrophages against the sporozoites of E. tenella was much higher in those isolated from bursectomized chickens (80%) than in those isolated from normal chickens. Because both bursectomized and normal chickens can be immunized by repeated infections, differences in the distribution among cloned T cells suggest different effector mechanisms of immunity against coccidiosis in these chickens. Lack of B cells seem to affect the development of T cell immunity as reflected by slower development of immunity and enhanced activation of cytotoxic T cell function.

Anti-idiotypic antibody with potential use as an Eimeria tenella sporozoite antigen surrogate for vaccination of chickens against coccidiosis.

Anti-idiotypic antibodies were raised in rabbits against four monoclonal antibodies with specificity for the surface antigenic determinants of Eimeria tenella sporozoites, the infective stage of the coccidial parasite. Two of the monoclonal antibodies (1073 and 15-1) transferred passive protection in chickens against E. tenella infection. The polyclonal anti-idiotype antibody preparations against protective monoclonal antibodies contained specificities for the paratope-associated idiotypes of these monoclonal antibodies, as assessed by the competitive inhibition of binding of the homologous idiotype-anti-idiotype by the sporozoite antigen. Competitive inhibition of binding of homologous idiotype-anti-idiotype by the parasite antigen was not observed when the anti-idiotype antibody preparations against monoclonal antibodies 1546 and 1096 were tested. The anti-idiotype 1073 and 15-1 antibodies functioned as surrogate antigens in vivo when used for vaccination of young chickens, as evidenced by the induction of partial protective immunity against subsequent challenge infection with virulent parasites and induction of antisporozoite antibodies. These data clearly support the view that anti-idiotypic antibodies raised against the paratope-associated idiotypes can mimic pathogen antigens and therefore can provide a possible alternative approach for the vaccination of chickens against coccidiosis.

Parasite exposure elicits a preferential T-cell response involved in protective immunity against Eimeria species in chickens primed by an internal-image anti-idiotypic antibody.

Polyclonal anti-idiotype 1073 (anti-Id 1073), raised against a monoclonal antibody specific for the protective epitope(s) of Eimeria tenella sporozoites, induced cell-mediated immune (CMI) responses in bursectomized chickens. Whereas alhydrogel-adsorbed anti-Id 1073 was sufficient to engender the CMI response at 4 h after injection, induction of the CMI response at 24 h required both alhydrogel and muramyl dipeptide sterol. Exposure of immunized chickens to live parasites prompted a dichotomous effect on the CMI response engendered by anti-Id in that the 4-h CMI response was preferentially stimulated and the 24-h CMI response was down regulated. Both types of CMI response were transferable to naive chickens by T cells from anti-Id 1073 immune donors or by parasite-specific T cells from clones 21 and 27. These T-cell clones were generated from chickens immunized by repeated infections with E. tenella and showed in vitro proliferative responses to anti-Id 1073. The abilities of T cells from clone 21 to selectively transfer the 4-h CMI response and to generate gamma interferon to activate macrophages for their cytotoxic effects on Eimeria sporozoites correlate with the preferential stimulation by parasites of the 4-h CMI response in chickens immunized with anti-Id 1073. These data show that anti-Id 1073 mimics the protective epitope(s) of the parasite and primes chickens for protective CMI responses. Cytotoxic T cells, equivalent to the mammalian T-cell subset of the Lyt2+ phenotype, appear to be the primary effector T cells in the CMI response engendered by anti-Id 1073 against Eimeria parasites.

Production of auto-anti-idiotype antibody during the normal immune response. XI. Ficoll-induced variations in auto-anti-idiotype production during the response to 2,4,6-trinitrophenyl-Ficoll.

The responses to 2,4,6-trinitrophenyl conjugates of different Ficoll preparations differ with respect to the magnitude of the accompanying auto-anti-idiotype (Id) response in both mice and chickens. Evidence is presented that reduced auto-anti-Id production in the chicken is due to the activation of suppressor activity by some preparations of Ficoll.

Physiology of IgD. V. Enhancement of antibody responses in vivo by allo anti-IgD is due primarily to an indirect effect on B cells.

Although responses of BALB/c mice to TNP-Ficoll or TNP-Brucella abortus are usually decreased by injection of allo anti-IgD (anti-Igh-5a) given 1 day before antigen, increased responses are obtained if a lymphokine mixture (SN) containing IL 2 is also injected. Simultaneous injection of anti-IgD and SN 4 days after priming with TNP-KLH induces an increase in antibody production similar to that induced by a second antigen injection. Injected together with a second injection of TNP-KLH at that time, anti-IgD and SN cause a synergistic enhancement of the secondary response. In allotype heterozygous (BALB/c X SJL)F1 mice injected with anti-IgD directed against one allotype, this enhancement of the secondary response is seen predominantly in the alternate allotype, because the IgG response of linked allotype specificity is slightly suppressed by the anti-IgD alone and is less enhanced than the alternate allotype by anti-IgD plus SN. Cells from unprimed heterozygous mice, incubated with anti-Igh-5a in vitro and transferred, together with antigen, to TNP-KLH-primed recipients, cause a much greater enhancement of the IgG responses of the Igb than of the Iga allotype in recipients. If, however, SN is also injected into the recipients, the anti-TNP response of both IgG allotypes is greatly enhanced.

Production of auto-anti-idiotype antibody during the normal immune response. X. Response to TNP-Ficoll in the chicken.

The spontaneous production of auto-anti-idiotype (Id) was demonstrated after injection of chickens with trinitrophenylated Ficoll (TNP-F) by: (a) the presence of hapten-augmentable plaque-forming cells (PFC), (b) the ability of serum and of hapten eluates from immune spleen cells to cause hapten-reversible inhibition of anti-TNP plaque formation, and (c) an enzyme-linked immunosorbent assay (ELISA). Tests for anti-Id using the ELISA and hapten-reversible inhibition of PFC correlated very well. As in the mouse, the incidence of hapten-augmentable PFC was reduced by thymectomy and increased by the transfer of TNP-F-immune spleen cells. Hapten-augmentable PFC were also observed during the immune response of chickens to p-azobenzene arsonate-conjugated Brucella abortus.

Immunoaffinity purification of avermectin-binding proteins from the free-living nematode Caenorhabditis elegans and the fruitfly Drosophila melanogaster.

Avermectin-binding proteins from the free-living nematode worm Caenorhabditis elegans and from the fruitfly Drosophila melanogaster were purified to homogeneity via a three-step procedure. The binding proteins were covalently labelled using a radioactive photoaffinity probe and then partially purified on a Sephacryl S-300 gel-filtration column. The radiolabelled binding proteins were then purified by immunoaffinity chromatography using a monoclonal antibody to avermectin covalently attached to Protein A-Sepharose beads. Three affinity-labelled Drosophila proteins with molecular masses between 45 and 50 kDa were isolated in this way and then separated from each other by electroelution. This three-step protocol provides a rapid technique for receptor purification which may be of use in the purification of other binding proteins.

Physiology of Igd. II. Lack of humoral immune responsiveness in lymph nodes of mice treated with anti-IgD from birth.

Previous studies have shown that anti-IgD-suppressed mice give normal primary and secondary splenic plaque-forming cell responses following i.v. challenge, although mice suppressed by the injection of anti-IgD from birth lack IgD-bearing cells in all lymphoid tissue examined. The present studies show that, in contrast, secondary immune responses in regional lymph nodes of such mice, even after i.v. priming with trinitrophenylated B. abortus, respond to a challenge injection in the footpad up to only less than 10% of control levels. When compared with respect to B cell numbers transferred, primed spleen cells from control and anti-IgD-suppressed mice are about equally effective in producing adoptive secondary plaque-forming cell responses in the spleens of recipient mice. Lymph nodes in recipients of anti-IgD-suppressed primed spleen cells show much lower responses than do lymph nodes in recipients of control primed cells, both upon immediate and delayed challenge with antigen in the footpads. It is concluded that the immunodeficiency caused by suppression with anti-IgD is much more marked in peripheral lymph nodes than in the spleen. The possible relationship of these results to the migratory properties of IgD+ as compared to IgD-B cells is discussed.