Allotype suppression in the chicken. IV. Deletion of B cells and lack of suppressor cells during chronic suppression

Embryonally induced allotype suppression in M-1,G-1 heterozygous chickens was stable for at least 18 months after hatching. Suppression was established rapidly since injection of antigen only 4 days after anti-IgM-1 antiserum failed to abrogate its effect. Injected chickens had undetectable serum levels (i.e. less than 40 micrograms/ml) of the suppressed IgM-1 and low (0.3-0.6 mg/ml) levels of the linked IgG-1 allotype. This correlated with a complete depletion of cells bearing the relevant IgM-1 allotypes and a compensatory increase in the alternative nonsuppressed IgM-1 allotype-bearing cells in the spleen, peripheral blood and bursa. Cell transfer studies suggested that suppression could not be attributed to allotype-specific suppressor cells.     

Allotype suppression in the chicken. I. Generation of chronic suppression in heterozygous but not in homozygous chickens.

The injection of antibody directed against either the IgM-1 a or IgM-1 b allotype into heterozygous (M-l^a/M-l^b, G-l^g/G-lⁱ) B 14-line chickens produced suppression of the relevant IgM-1 and genetically linked IgG-1 allotypes in their serum, as quantitated by single radial immunodiffusion. Suppression of the IgM-1 and IgG-1 allotypes was associated with a compensatory increase in the alternative IgM-1 and IgG-1 serum allotype levels. This suppression was induced (a) by passive injection of anti-allotype antiserum into 13-day-old embryonal or neonatal recipients, or (b) by egg yolktransmitted antibody in chickens hatched from homozygous B 14 A (M-l^a, G-l^g) hens immunized against the IgM-l b of the homozygous B14C (M-l^b, G-lⁱ) rooster. Heterozygous chickens injected embryonally with anti-allotype antiserum were profoundly suppressed for at least 16 weeks after hatching, while neonatally injected chickens showed a gradual recovery of both IgM-1 and IgG-1 allotypes over the same period. Suppression of the IgM-1 b allotype could be induced in heterozygotes which had inherited the M-l^b allele either maternally or paternally. However, no suppression of either IgM-1 or IgG-1 levels could be detected in homozygous chickens injected with the relevant anti-allotype antiserum. Hence, allotype suppression only occurred in M-1 heterozygous chickens which had an alternative source of B cells available. The involvement of a B cell surveillance mechanism in allotype suppression is postulated and the possible role of suppressor cells is discussed.     

Allotype suppression in the chicken II. Suppression in homozygous chickens with antiallotype antibody and allotype-disparate B cells

Injection of heterozygous (M-1³/M-1^b, G-1^g/G-1ⁱ) B 14-line chickens with antisera directed against either IgM-1a or IgM-1b induced suppression of the relevant IgM-1 and genetically linked IgG-1 allotypes, whereas a mixture of anti-M-1a and anti-M-1b antibodies failed to produce allotype suppression. Injection of anti-M-1 antiserum into M-1 homozygous chickens induced only a transient delay of a few days in the appearance and rise of serum IgM-1 levels. However, suppression of host allotypes was induced by injecting M-1, G-1 homozygous neonatal or embryonal recipients with anti-M-1 antisera together with B locus-histocompatible allotype-disparate spleen, bone marrow or bursal cells. The active cell type were donor B cells, which established chimerism in the injected hosts, whereas peripheral blood T lymphocytes from agammaglobulinemic donors were ineffective. Allotype suppression was attributed to a homeostatic control mechanism which is exerted by normal B cells (but not T cells) over B cell recruitment in anti-M-1 antibody-treated, immature hosts.     

Effects of IgM Allotype Suppression on Serum IgM Levels,B-1 and B-2 Cells,and Antibody Responses ir Allotype Heterozygous F1 Mice

IgM allotype heterozygous F1 mice were independently suppressed for Igh6a or Igh6b to evaluate the contribution of B-1 and B-2 cells to natural serum IgM levels and Ab responses. B-2 B cells expressing IgM of the suppressed allotype were evident in the spleens of suppressed mice 4 to 6 weeks after cessation of the suppression regimen, whereas B-1 B cells of the suppressed allotype were undetectable for up to 9 months. Although serum IgM of the suppressed allotype was initially depleted in mice suppressed for either allotype, by 7 months of age, there were detectable levels of IgM of the suppressed allotype in the serum; however, the levels were significantly below that found in nonsuppressed mice. When mice were immunized with either the T-independent or T-dependent form of phosphorylcholine, those suppressed for either allotype, and consequently depleted of B-1 B cells of that allotype, did not respond with phosphorylcholine-specific IgM of the suppressed allotype. In contrast, when mice were immunized with α1-3 dextran, the Igh6a allotype-suppressed mice were able to produce dextran-specific IgM of that allotype. These results show that allotype-bearing B-1 cells of both allotypes can be effectively suppressed by this suppression protocol and this produces long-lasting effects on B-1 cell levels and serum IgM of the suppressed allotype. These observations reflect the derivation of the majority of B-1 cells from fetal-neonatal precursors, which cannot be replaced by newly emerging B-2 cells of adult origin. Their ablation by antibody treatment results in permanent alterations to the adult B-cell repertoire.     

Antibody-mediated allotype suppression in adult mice: the role of antigen, effector isotype and regulatory T cells

It has been reported (Contemp. Top. Immunobiol. 1974. 3:41) that allotype-specific T suppressor cells can be induced after monoclonal anti-allotype treatment of neonatal (BALB/c X SJL)F1 (Igha/b) mice. Here we show that (BALB/c X CB20)F1 adult-derived spleen cells (SC) are, by contrast, potently suppressed by monoclonal allotype-specific reagents, (when transferred into irradiated BALB/c recipients) in the absence of primary T suppressor cell induction. Such suppression is only induced in activated B cells [exposed to lipopolysaccharide or sheep red blood cells (SRBC)], and is probably dependent on the isotype of the anti-allotype sera administered. For example, two independently produced IgG1 monoclonal reagents raised against the Igh-1b allotype were poorly suppressive or nonsuppressive, whereas an IgG3 and an IgG2a monoclonal antibody induced a 90% suppression of the target allotype in transferred adult SC. It was found that suppression was not due to a depletion of antigen-specific T cell help since: (a) the addition of SRBC-educated T cells did not break suppression and (b) suppressed SC were as good a source of T cell help as normal SC, in the response of virgin or memory B cell (Thy-1-depleted) responses to SRBC in vivo. Suppression was maintained in suppressed cells which had been rechallenged with SRBC after transfer into a second irradiated recipient, but was not induced in normal SC when these were admixed with an equal number from this suppressed SC population. These findings point to a possible mechanism for the regulation of B cell expression, through the formation of an antibody-Ig receptor complex at the surface of the B lymphocyte. After complexing the target cell is either deleted or inactivated. The response to SRBC was reduced or ablated for at least 70 days after treatment with a single dose of anti-allotype serum.     

Allelic exclusion of M1 (IgM) allotype on the surface of chicken B cells.

The membrane expression of M1 (IgM) and G1 (IgG) allotype markers on peripheral blood lymphocytes was examined by immunofluorescence. In homozygous chickens 15% lymphocytes stained with either anti-M1 or with polyspecific rabbit anti-Ig serum, suggesting that M1 is expressed on the surface of probably all B lymphocytes. In heterozygous M1a/M1b individuals antisera against either allelic antigens reacted with 50% of the total surface Ig positive cells as evidence for the allelic exclusion of surface M1 expression. Donor allotype synthesis was determined from serum allotype levels in congenic cyclophosphamide-treated recipients of lymphoid cells. Treatment of cells with anti-M1 serum prior to transfer inhibited both M1 and G1 allotype synthesis. The treatment of heterozygous (M1a/M1b, G1a/G1e) cells with anti-M1b serum inhibited specifically the synthesis of M1b and G1e allotypes controlled by linked genes from the same parental chromosome. Thus, B cells which had been the targets for anti-M1 antibody mediated suppression are also subject to allelic exclusion.     

Escape from b locus allotype suppression in the rabbit is mediated by IgM molecules bearing an allotope-restricted variant of kappa light chain

Rabbits homozygous for b6 at the kappa light chain b locus were suppressed for the expression of the b6 allotype and then induced to produce auto anti-b6 antibody. Rabbits which subsequently escaped suppression produced auto antibody with restricted allotype specificity. Escape from allotype suppression was mediated by IgM bearing a kappa chain variant with a restricted number of b6 allotopes and having a diminished interaction with auto anti-b6 antibodies from the same and other rabbits escaping b6 suppression. This suggested that there were allelic variants or subpopulations of the b6 light chain which were under independent regulation of expression, clearly influenced by the specificity of auto anti-allotype antibody. Since escape from suppression was mediated by IgM it is proposed that a normal pathway of B cell differentiation occurs during recovery from suppression.     

Tolerance to allotypic determinants induced by lymphoid cells from congenic mice bearing the allotype.

Mice of one allotype (Igb) were immunized against immunoglobulin from a congenic strain of mice bearing another allotype (Iga). This Igb anti-Iga response was profoundly suppressed by injecting lymphoid cells from congenic Iga mice but not by serum Iga. Suppression was specific, could be induced by congenic B cells but not histoincompatible lymphoid cells and depended both on the time of administration of cells relative to immunogenic challenge and to the number of cells injected. Unresponsiveness was more easily induced in neonates than adults. It is considered that tolerance to the allotype depends on the properties of the cells to which they are bound.     

Expression of b 4 and b 5 chi light chain allotypes by B and pre-B cells in allotype-suppressed and neutralized b4b5 rabbits.

In previous studies, we described a primitive lymphoid cell found in fetal liver and in the bone marrow of older rabbits which contained cytoplasmic IgM but lacked surface IgM detectable by immunofluorescence. In heterozygous b⁴b⁵ rabbits, the pre-B cells in which we could detect these kappa chain allotypes appeared to exhibit allelic exclusion. In the present study, we investigated the effects of allotype suppression and its neutralization on the expression of the b4 and b5 allotypes by B and pre-B cells from the spleens and bone marrow of b⁴b⁵ rabbits. We found that in young allotype suppressed rabbits, pre-B cells of the suppressed allotype persist in bone marrow when B cells of the suppressed allotype are absent or severely depleted. The persistence of pre-B cells of the suppressed type supports the view that pre-B cells differ in their responsiveness to external influences such as anti-Ig compared to B lymphocytes. Injection of serum with b5 immunoglobulin into b⁴b⁵ animals suppressed 14-23 days previously for b5 was followed by the appearance of increased proportions of b 5 B cells in spleen within 24 h. Surviving pre-B cells are a likely source of these rapidly appearing B cells as well as of the B cells bearing surface immunoglobulin of the suppressed allotype which appear during the recovery phase of allotype suppression.     

In vitro studies of allotype suppression in mice

Long-term allotype suppression in (SJL x BALB/c)F₁ mice has been investigated in vitro, using a culture system which can be maintained over a period of at least 2 weeks. Spleen cells from (SJL x BALB/c)F₁ nonsuppressed mice, primed 2 to 5 months earlier, were cultured at a concentration of 3 x 10⁶ cells together with 10⁶ spleen or lymph node cells from suppressed or from nonsuppressed (control) mice and challenged in vitro with SRBC. Cultures were assayed on days 5, 8, 11, and 14 by the PFC assay, using specific anti-allotype sera to develop indirect plaques. Cells from suppressed mice were extremely efficient in preventing a “b” allotype response of the primed cells in vitro, even though the “a” allotype response in the same cultures was unaffected. A time lapse of approximately one week in culture was required before suppression was very obvious. The suppressive effect was abolished by treatment of suppressor cells with anti-θ serum. Evidence is presented that the suppressive effect is due to the production of a diffusible factor, rather than to a direct cell-cell interaction.     

Regulation of allotype expression in heterozygous rabbits. II. Concomitant suppression of b4 and b6 allotypes in the same cell.

Cells from heterozygous b4b6 rabbits were treated at 4 degrees with anti-b4 or anti-b6 antibodies and then warmed at 37 degrees. A disappearance of both b4 and b6 allotypes (concomitant modulation) ensued. When cells which had undergone extensive comodulation were cultured overnight we noted that those cells were unable to re-express either allotype at pre-modulation levels. This suppression was likely linked to the initial events which culminated in comodulation. Those cells were not further suppressible when suppressive antibodies were added to the cultures whereas cell cultures which had undergone little or no previous modulation or comodulation were readily suppressed for both allotypes after anti-allotype antibodies had been added to the cultures overnight (concomitant suppression). This indicated that in vitro suppression of allotype may depend on cell surface allotype being present at a sufficiently high density. We present data which show that events at the cell surface may play a role in the regulation of cell surface allotype expression and propose that concomitant suppression may have bearing on cellular mechanisms which control allotype expression and also allotype suppression.     

Cellular basis of an auto-anti-allotypic mechanism for the maintenance of chronic allotype suppression in the rabbit.

Immunocytochemical identification of antibody-forming cells (AFCs) in situ was used to test the hypothesis that the maintenance of chronic allotype suppression in heterozygous rabbits is the result of an autoimmune B-cell-mediated response. Appreciable numbers of B cells with antibody activity directed against the suppressed allotypic determinant were found in spleen and bone marrow sections of all chronically suppressed rabbits examined. Appropriate double-staining was used to determine that such cells were of the non-suppressed allotype. These cells were indistinguishable from anti-allotypic AFCs found in larger numbers in spleens of normal heterozygous rabbits that had been immunized against a heterologous allotypic determinant. Auto-anti-allotypic AFCs were not found in suppressed rabbits less than 8 week old, nor were they found in normal (non-suppressed) heterozygous rabbits or chimeric rabbits formed by the injection of histocompatible but allotype-mismatched lymphoid cells at birth. The findings reported here support the hypothesis that the long-term maintenance of allotype suppression in the rabbit may result from the suppressive activities of autoimmune B cells. It is suggested that the suppression of an allotype during the first few weeks of life could result in a loss of tolerance to a self-determinant. The kinetics of auto-anti-AFC production support this idea in showing that such cells are generated following the decline of the antibody used to induce suppression. The triggering event may be the emergence of B cells expressing the previously suppressed gene product.     

Analysis of the immunoglobulin isotype expression by peripheral blood rosette forming cells in chickens

We have analysed the nature of rosette forming cells (RFC) in peripheral blood lymphocytes (PBL) from chickens which had been immunized with SRBC 7 days previously. Although none of the RFC secreted antibody immediately after harvest, a PFC response was detected after in vitro culture in the presence of SRBC. This response was of IgG isotype and was abolished by depleting RFC from cell suspensions prior to culture. In chickens with a partial immunodeficiency (produced by bursectomy 3 days post-hatching) RFC counts and IgG antibody levels were suppressed during the primary response but the in vitro memory response was unimpaired when tested 4 weeks later; thus, RFC appear to be the precursors of antibody secreting but not memory cells. The normal levels of IgM haemagglutinins found in bursectomized chickens suggested that peripheral blood RFC constitute part of the maturation pathway of IgG but not IgM antibody producing cells.

Incubation of primed PBL with anti-M1 (IgM) allotype sera inhibited rosette formation. With PBL from M1^a/M1^b heterozygous chickens, only 50% RFC inhibition was achieved by either anti-M1^a or anti-M1^b serum. This result, when interpreted in terms of allelic exclusion, implies that M1 receptors are endogenous cell products rather than passively acquired molecules. We conclude that RFC in chicken PBL represent B cells which are committed to IgG antibody synthesis and still express a high density of IgM antigen binding receptors at an advanced stage of maturation.

     

Allotype suppression induced in the adoptive transfer system: the variables of the system and an apparent absence of a role for T cells.

A study has been made of the variables concerned in allotype suppression of adult spleen cells in the adoptive transfer system. These are; SRBC (antigen) dose; the dose and timing of injection of anti-allotype serum IgG; the number of spleen cells transferred and whether these cells were taken from primed or unprimed donors. Adoptively transferred primed cells are considerably less susceptible to suppression by concomitantly injected anti-allotype serum IgG than are unprimed spleen cells. Injection of anti-allotype serum during the period after adoptive transfer, has shown that primed cells loose their susceptibility sooner (2 days) than the unprimed cells (4 days). Allotype heterozygous CBA spleen cells are less susceptible heterozygous CBA spleen cells are less susceptible to allotype suppression than either allotypically homozygous or heterozygous non-H-2k cells (H-2b,d, or s). Allotype suppression of the TI IgG response to DNP-Ficoll was measured 7 days after adoptive transfer of allotype-homozygous cells from both normal and nude CBA mice (unprimed). The results indicate that T cells do not play a role in the initiation of short-term allotype suppression in the adoptive transfer system.     

Igh-1b-specific CD4+CD8- T cell clones of the Th1 subset selectively suppress the Igh-1b allotype in vivo.

The demonstration of major histocompatibility complex (MHC)-restricted T helper (Th) cells specific for peptides from the variable (V) regions of syngeneic immunoglobulin (Ig) (idiopeptides) opens the possibility that Th cells regulate B cell functions via idiopeptide-based cognate T-B interactions. As a model for such interactions we investigated the influence of Ig allotype-specific T cells on the differentiation of H-2-syngeneic B cells expressing that particular Ig allotype. We established a BALB/c (H-2d, Iga) CD4+CD8- T cell line and clones of the Th1 subset (interleukin 2+, interleukin 4-, interferon-gamma+, tumor necrosis factor-alpha+) that recognized Igh-1 (IgG2a) of the b allotype (Igh-1b) together with I-Ad. These T cells specifically suppressed surface Igh-1b+ B cells in vitro and in vivo. In 12 out of 15 6-week-old (BALB/c X B10.D2)F1 mice neonatally injected with Igh-1b-specific T cells, the serum Igh-1b concentrations were less than 5% of the levels in the controls. Thus, allotype suppression can be accomplished solely by adoptive transfer of Igh-1b-specific CD4+ T cells. The in vivo suppression was specific for Igh-1b+ B cells as the recipients' levels of Igh-1a and Igh-4b (IgG1b) were unaffected. The V beta 14-specific anti-T cell receptor (TcR) monoclonal antibody 14-2 inhibited activation of hybridomas derived from two of the clones. Collectively the data indicate that suppression resulted from cognate interactions between allopeptide-specific TcR alpha/beta+ T cells and normal unmanipulated B lymphocytes presenting their endogenous Igh-1b in association with MHC class II molecules. The data support the possibility that normal B cells can be suppressed by idiopeptide-specific T cells in vivo.     

Epitope-specific regulation. III. Induction of allotype-restricted suppression for IgG antibody responses to individual epitopes on complex antigens

Maternal antibody to the paternal Igh-1b (IgG_2a) allotype induces chronic suppression for Igh-1b (1b) production in (BALB/c × SJL)F₁ hybrid mice. These mice characteristically remain incapable of producing lb antibody responses until about 3 months of age and then enter a remission phase during which they produce normal 1b antibody responses to antigens introduced initially at this time. Thus young allotype-suppressed mice do not produce lb antibody responses to dinitrophenylated keyhole limpet hemocyanin (DNP-KLH) and 1b anti- KLH. The suppression of 1b antibody responses in young allotype-suppressed mice prevents the expression, rather than the development, of 1b memory for priming antigen epitopes. Furthermore, it not only prevents the expression of such memory cells initially but results in the induction of a continued suppression that specifically prevents their expression after the onset of remission. Thus mice primed with DNP-KLH while allotype suppression is still active develop normal 1b memory for DNP and KLH but nonetheless fail to produce lb anti-DNP and 1b anti-KLH responses, even when restimulated with DNP-KLH during remission. These mice also fail to produce 1b anti-DNP when stimulated with DNP on an unrelated carrier molecule, i.e., with DNP-chicken gamma globulin (CGG). This suppression is both epitope-specific and allotype-specific. That is, although 1b responses to DNP on CGG are suppressed, 1b responses to CGG epitopes on DNP-CGG proceed normally. Furthermore, there is no suppression of other isotype and allotype responses either to DNP or to the CGG epitopes. These data therefore define an Igh-restricted epitope-specific mechanism that can be induced to persistently suppress 1b antibody responses to epitopes introduced initially during active (1b) allotype suppression.     

T-Cell suppression by cyclosporin-A enhances infectious bursal disease virus infection in experimentally infected chickens

In this study, the role of T lymphocytes was investigated in chickens experimentally infected with infectious bursal disease virus (IBDV). Chickens were treated with cyclosporin-A (CS-A), a selective T-cell suppressant drug, by the intramuscular route, starting 3 days before virus infection and every third day thereafter,and infectious bursal disease pathogenesis was compared in such T-cell suppressed and intact chickens using a vaccine strain; namely, Georgia and a field isolate of IBDV. Treatment of chickens with CS-A caused a significant suppression of phytohaemagglutinin-A specific proliferative responses of peripheral blood mononuclear cells. The virus neutralizing antibody titres in such CS-A treated chickens were not suppressed. T-Cell suppression resulted in an increase in the severity of gross lesions caused by IBDV and extensive muscular haemorrhages were observed in such chickens between 15 and 21 days post-inoculation. Similarly, there was a marked increase in the severity of infectious bursal disease-specific microscopic lesions in the bursa of T-cell suppressed chickens. Consistently higher titres of virus were observed in bursa of CS-A treated chickens. Virus titres were 1 to 2 log10 higher in the T-cell suppressed chickens as compared with the intact ones. These studies suggest that T cells play a role in limiting the IBDV infection.     

Allotype suppression of the responsiveness of adoptively transferred adult spleen cells induced by affinity prepared anti-allotype antibody.

In an adoptive transfer assay it has been shown that a form of allotype suppression can be induced in adult spleen cells. Use of SPF congenic mice, and either anti-allotype serum freed of IgM rheumatoid factor or affinity prepared antibody, has helped to eliminate the possibility of a role for non-specific factors in allotype suppression. Both memory-cell development and a conventional primary response are more susceptible to suppression than memory expression in a secondary response. Non-specific suppressive effects on isotypes other than that of the target allotype (CH) are present but are much smaller than for specific suppression.     

A T-cell subpopulation committed to help B cells for immune responses restricted to IgM type.

Immune responses againt bovine serum albumin by chicken were dependent upon thymus-derived cells. Thirty-five of seventy chickens that had been neonatally thymectomized and subsequently immunized with bovine serum albumin produced IgM antibodies, but not IgG antibodies, against the antigen. T cells (IgM-T cells) of such chickens were able to help B cells to produce IgM antibody responses but were not able to help them to switch IgM- to IgG-antibody responses. Helper activity of the IgM-T cells was much less susceptible to the cytotoxic effect of anti-thymus cell serum and complement than was that of normal T cells. The introduction of the IgM-T cells into normal chickens at the same time as the initiation of immunization of the chickens did not affect immune responses by them at all, indicating the absence of suppressor T cells in the IgM-T cell preparations. Injection of chicken thymus factor into immunodeficient chickens transplanted with normal B cells and IgM-T cells developed the capability to help B cells to switch IgM- to IgG-antibody responses. On the basis of these findings the authors propose the existence of helper T cells which are characterized by peripheralization in early periods of ontogeny, the possession of helper activity for only IgM-antibody responses, the lack of helper activity for the switch from IgM- to IgG-antibody responses and relative insusceptibility to the cytotoxic effect of anti-thymus cell serum and complement.     

Carrageenan-induced suppression of chicken lymphocyte proliferation

Mitogenic responsiveness of peripheral blood lymphocytes (PBL) of chickens was suppressed by either pretreatment with or addition to the culture medium of various concentrations of carrageenan (CGN). Pretreatment for 1 hr significantly suppressed response to Concanavalin A (Con A) and Pokeweed mitogen (PWM) but did not affect Phytohemagglutinin (PHA) induced stimulation. Extension of the pretreatment period to 4 hrs suppressed response induced by all three mitogens. On the other hand, addition of carrageenan to the culture medium caused a dose-dependent suppression of PHA and Con A mediated response, but the effect on stimulation due to PWM was equivocal. In addition, low concentrations of CGN were weakly mitogenic to PBL and splenic lymphocytes.