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.

     

Rosette plaques with lymphoid cells from heterozygous rabbits. Ani-hapten antibody-forming cells displaying either one or both b locus surface allelic markers.

A rosette-plaque model was employed to test for the expression of b locus allelic markers at the surface of lymph node lymphocytes (LNL) from heterozygous (b4,6) rabbits, 5 days after immunization with NIP-diphtheria toxoid. Before immunization, in all animals examined, LNL displaying both b4 and b6 determinants at the surface (range 5-20 per cent) were detected, the remainder consisting of cells exhibiting only one or the other determinant. After immunization, five of the thirteen heterozygotes apparently had gone into allelic exclusion as LNL from these animals showed only b4 or b6 rosettes which secreted anti-NIP antibody in the plaque. The eight remaining rabbits remained in allelic inclusion. Since cytophilic uptake of allotype might have contributed to double expression, LNL from immunized animals were treated with pronase to remove surface immunoglobulin. When the stripped cells were cultured overnight in serum-free medium, reappearance of b4, b6, and b4 plus b6 expressing cells was seen. When pronase-stripped cells were incubated in cycloheximide (20 mug/ml) for 5 hr, no allotype synthesis was found but inhibition was relieved when the cells were washed free of the antibiotic. Regrowth resulted in rosette levels similar to those found originally in the three allotype-bearing populations. Stripping the cell surface allotype with pronase, and allowing regrowth of allotype overnight also resulted in one of four animals regaining the ability to express both allotypes at the surface in the plaque-forming situation. Lymphocytes from homozygous controls (b4,4 and b6,6) displayed their own individual allelic markers either when the cells from each were tested alone or in combination, unimmunized or immunized. An additional finding was the apparent lack of allelic preference for NIP in the heterozygotes as approximately similar numbers of cells were found bearing the b4 and b6 marker at the surface in the NIP plaque.     

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.     

Homogeneity of antibodies produced by clones in vitro

Peripheral blood lymphocytes from primed rabbits were stimulated with sheep red cells (SRC) in microcultures. In this method, if a limiting number of lymphoid cells is used, only a small percentage of the cultures responds with production of antibody. We have analyzed the antibody produced in the responding cultures from the point of view of allotype and electrophoretic mobility. In rabbits heterozygous at the b locus, the anti-SRC antibody in single cultures was shown to express either one or the other allele. In a few of the cultures the antibody produced did not express any b locus allotype and was therefore assumed to be carrying lambda-type L-chains. In high voltage agar electrophoresis, the antibody activity of most of the positive cultures was localized in a single band, the position of which varied from culture to culture. These results show that the antibody produced in single microcultures is the product of single B cell clones. The homogeneity for allotype observed in the antibody produced by single microcultures is strong evidence that allelic exclusion, once achieved, is maintained throughout clonal proliferation.     

Allelic exclusion frequency analysis and molecular characteristics of immunoglobulins secreted by the hybridomas expressing both allelic genes

The investigation of 750 B-lymphocyte hybridoma clones obtained by fusion of mouse myeloma and newborn heterozygous Igk-1a/Igk-1b rat splenocytes has revealed that 9.8% of Ig kappa-chain loci are rearranged productively. Seventeen hybridomas secrete kappa-chains of both allelic variants. The analysis of IgM molecules of 9 such clones demonstrated that in 6 cases only one L-chain allotype, either 1a or 1b, is present in IgM. Thus for the first time the high frequency of selective association of H and L chains in Ig-producing cells was shown. Evidently this selectivity may function as one of allelic exclusion mechanisms at the Ig assembly stage.     

Avian B cell precursors: surface immunoglobulin expression is an early, possibly bursa-independent event

The avian bursa of Fabricius contains about 1 X 10(4) discrete follicles, each of which is colonized by a small number of lymphoid progenitor cells during embryonic life. We have previously shown (J.R.L. Pink et al., Eur. J. Immunol. 1985. 15:617) that all, or almost all B cell progenitors in the bursae of 4-day-old chicks express cell surface IgM. In this report, we have analyzed the distribution of cell surface (s)IgM-1 allotypes within individual follicles of (M-1a/M-1b) allotype heterozygous birds. Although the majority of follicles contained a mixture of sIgM-1a+ and sIgM-1b+ cells, a significant proportion of isolated follicles contained exclusively sIgM-1a+ or sIgM-1b+ cells. Statistical analysis of the frequency of such "M-1a" and "M-1b" follicles demonstrated that the sIg+ B cells in the bursae of 4-8-week-old birds are derived from 2-4 allotypically committed precursor cells per follicle. Since we have previously shown that each bursal follicle is colonized by 2-5 pre-bursal stem cells, these cells must be committed to the eventual expression of one or other allotypic haplotype before they have undergone extensive proliferation within the bursa. In addition, we show that almost all B progenitor cells from the bursae of chicks which had been allotype suppressed as embryos were committed to synthesis of the nonsuppressed allotype, showing that this commitment was essentially complete at the time of suppression (i.e. before 19 days of incubation). Finally the bone marrow of 16-day embryos was used to reconstitute the bursal lymphocytes of cyclophosphamide-treated host embryos. Reconstitution was inhibited by anti-Ig antiserum indicating that most 16-day embryonic BM-derived bursal cell precursors also express sIgM. These results raise the possibility that expression of sIgM may be controlled by a "biological clock" rather than by any inductive capacity of the bursal microenvironment. Furthermore, these results provide further evidence that in normal birds a self-renewing sIg+ B cell population in the hatched chicken is the sole source of B cells in the adult.     

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.     

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.     

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.     

Induction and regulation of silent idiotype clones Idiotype regulation

In order to study whether allotype linkage of idiotype expression is related to the total absence of structural gene(s) or to regulatory mechanisms acting on its phenotype expression, the M460 clone expression was followed in B cells from various inbred and recombinant strains of mice cultured at low density in the presence of lipopolysaccharide. Under these conditions, all cultures were found to contain anti-trinitrophenyl (TNP) antibody plaque-forming cells, some of which were inhibitable by anti-M460 immunoglobulins. In the course of these studies, it also became apparent that, even in the absence of T cells, B lymphocytes are able to exert regulatory functions. Experiments carried out using anti-M460 hybrid cells F6 (51), and affinity-purified anti-(anti-M460) antibodies revealed that clonotype regulation by B cells is mediated by idiotype-auto-anti-idiotype interactions.     

Is there really a “lack of natural tolerance to allotypic γ-globulins in rabbits”?

We present data of extended studies on the possibility of maternally derived allotype Ig inducing a state of natural immunological tolerance to a non-inherited allotype in the offspring. Rabbits homozygous at the a locus, encoding allotypes in the variable region of immunoglobulin heavy chains, and rabbits homozygous at the unliked b locus, encoding allotypes of the constant region of kappa 1 chains, were immunized at the age of 2 months against the non-inherited allotype of their heterozygous mothers to which they had been exposed in utero and in early life. As control, we immunized rabbits of the same Ig phenotype but born to homozygous mothers, and therefore not exposed to that allotype. Immunization was done in a3/a3 offspring of either a1/a3 or a3/a3 mothers, by injecting a 1 IgG, and in b6/b6 offspring of b4/b6 or b6/b6 mothers, by injecting b4 IgG. The IgG was injected either in a soluble form or emulsified in adjuvant. Injection of soluble IgG elicited only a low response, if any, revealing no differences between the various groups. All rabbits responded upon immunization with IgG in adjuvant. We have not found any good evidence for natural tolerance to a non-inherited allotype, although progeny of a1/a3 mothers had slightly decreased responses to a1. On the contrary, progeny of b4/b6 mothers responded even better than offspring of b6/b6 mothers, upon such immunization with b4. To induce tolerance experimentally, we injected newborn rabbits, either from heterozygous a1/a3 or from homozygous a3/a3 mothers, with a1 serum or IgG. Newborn of heterozygous b4/b6 or of homozygous b6/b6 mothers were injected with b4 serum or IgG in the same way. Such treatment resulted in partial tolerance to each allotype. In an attempt to amplify the tolerizing effect of the maternal a1 Ig, we injected newborn rabbits of a1/a3 mothers with the serum of their mother. The response upon subsequent immunization with a1 allotype of another individual did not differ significantly from the response of control rabbits. The response was much poorer when rabbits were injected with nonmaternal tolerogen at birth, and when the same Ig preparation was used as immunogen. In a control experiment, neonatal injection of xenogeneic proteins, human IgG or bovine serum albumin, clearly resulted in tolerance. We speculate that tolerance to allotypes is established in the T cell repertoire only but bypassed by recognition of idiotypic determinants on antigen molecules by helper T cells, which trigger anti-allotype antibody formation by allotype-specific B cells. The end result of it is a lack of natural tolerance.     

Long-term antibody synthesis in vitro. VI. Anti-allotype sera as probes of clonal products in affinity maturation.

A new experimental system is described for measuring the allotypic product of rabbit B cells during long-lasting in vitro antibody responses. The immunoenzymatic assays described allow determination of several parameters mapping in different regions of the same molecule, which can be measured and combined to yield a multidimensional picture of the time-course dynamics of antibody synthesis. The rabbit immune system responding to Escherichia coli beta-D-galactosidase was sample and disassembled by (a) culturing lymph node microfragments and (b) sorting out from among all anti-enzyme antibodies only those activating a mutant enzyme, AMEF, which bore the b4 or b9 allotype. A considerable simplification of the response was achieved in the microcultures as documented by cultures of heterozygous cells which produced only one allotype and by the fact that each culture showed a distinctive pattern when antibody titre, association constant, heterogeneity index, L-chain type, and k-chain allotype were considered together. This array of patterns was not an artifact but the result of disassembling a representative sample of the rabbit immune system into small components, since the b4/b9 ratio obtained by averaging the results of all cultures from a heterozygous rabbit lymph node was the same as the serum ratio. Despite the Poisson distribution of the responder microcultures, none of them was monoclonal; i.e. no antibodies homogeneous by all parameters tested were observed, This finidng supports the notion that in normal lymphoid tissue in its native tridimensional arrangement, one T cell can trigger several B cells clustered in one antibody-forming unit. This natural arrangement would ensure the monospecificity of the cluster (dictated by the T cell) while allowing for variation in affinity (depending upon the array of B cells in the unit). Accordingly our findings would results from the fact that as the size of the microfragments was reduced, the cells diluted out first were T cells, but as long as one of them was present, several B-cell clones were triggered. The b4/b9 pattern of any given culture remained constant over several months, but the ratio kappa/lambda underwent changes. An increase in molecules with non kappa-chains (which could not be reacted with anti-kappa-chain allotype antisera) was usually associated with a parallel decrease in antibody affinity. This occurred by the end of the antibody cycle and might be related to the regulation of antibody synthesis by T-cell suppressor factors.     

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.     

An alpha2-macroglobulin associated factor produced by T lymphocytes which provides polyclonal stimulation of B lymphocytes to maintain the turnover of their surface Ig.

The supernatant of 'crowded' but not 'spread' rabbit spleen cell cultures contains a macroglobulin factor which behaves in an Ig-turnover assay as any T-independent antigen or polyclonal B-cell activator (PBA). In the supernatants of crowded rabbit lymphoid cell cultures prepared in serum free medium, the factor was found to be associated entirely with the alpha-macroglobulin (alpha M) fraction (alpha 1 + alpha 2). This alpha M was most probably actively secreted by the lymphocytes because: (i) sequential supernatants obtained in serum free medium of crowded cultures contained equal amounts of alpha M as well as equal PBA activity; (ii) the alpha M became labelled when the cells were grown in medium containing a radioactive amino acid. Macrophages were not required for the production of PBA. PBA was not produced when either crowded B or T cells were cultured alone but only when they were cultured together. Purified T cells were not triggered by any plant lectin to produce PBA. By use of anti-alpha 2M allotype antibodies and B and T cells from different rabbits, the PBA was shown to have the allotype of the T-cell donor. The PBA was associated with rabbit alpha 2M but not alpha 1M. We concluded that upon close contact, B cells stimulate T cells to produce a PBA associated with alpha 2M.     

Immunoglobulin-specific T-B cell interaction. I. Presentation of self immunoglobulin determinants by B lymphocytes

Immunoglobulin (Ig)-specific T-B cell interactions have been studied in the model of T cell recognition of the kappa chain Ig kappa-1b allotype in Ig kappa-1-congeneic rat strains. An efficient presentation of endogenous Ig allotypic determinants by irradiated spleen cells from (WAG.1b x August)F1 (RT-1u/c; Ig kappa-1b/1a) rats to Ig kappa-1b-specific lymph node T cells from Ig kappa-1-congeneic (WAG x August)F1 (RT-1u/c; Ig kappa-1a) rats was demonstrated. This presentation was found to be sensitive to high irradiation doses (greater than 1000 rad). By fractionation of Ig kappa-1b+ F1 spleen cells on Percoll density gradient we have shown that a radioresistant, low-density fraction, consisting mainly of macrophages (M phi) and dendritic cells, triggers only weak Ig kappa-1b-specific T cell response. The high level of response was observed against radiosensitive spleen cell fractions of intermediate and high density, suggesting that B cells were the main antigen-presenting cells (APC) of Ig kappa-1b determinants of endogenous Ig. This conclusion was confirmed in the experiments using purified B cells from Ig kappa-1b-bearing rats. Earlier we have shown that the responsiveness of August (RT-1c; Ig kappa-1a) and WAG (RT-1u; Ig kappa-1a) rats to Ig kappa-1b in vivo is controlled by the dominant allele of an RT-1-linked Ir gene. August and (August X WAG)F1 rats were found to be responders to Ig kappa-1b while WAG rats were nonresponders. The same pattern of Ir gene-controlled reactivity was demonstrated using an Ig kappa-1b-specific T cell proliferation assay. Ig kappa-1b-specific F1 T cell response was only observed when Ig kappa-1b+ B cells or IgG (Ig kappa-1b)-pulsed M phi-bearing responder major histocompatibility complex (MHC) haplotype were used as the APC. Anti-RT-1 monoclonal antibody inhibition studies suggested that the RT-1Bc molecule is the main restricting element of T cell recognition of Ig kappa-1b+ B cell as well as exogenous IgG (Ig kappa-1b). We have demonstrated allelic exclusion of Ig kappa-1b presenting function by negatively and positively selecting for Ig kappa-1b+ and Ig kappa-1a+ B cells from heterozygous F1(Ig kappa-1b/1a) rats. This clearly indicate that the B cells presented exclusively Ig kappa-1b allotypic determinants of their own Ig.(ABSTRACT TRUNCATED AT 400 WORDS)     

Maternal allotype dominance and allelic exclusion in the B lineage cells of the newborn rabbit.

Newborn rabbits from parents which differ at the b allotype locus (kappa chain), show a strong maternal allotype dominance, especially in the bone marrow. This concerns not only the membrane but also the cytoplasm, and it cannot be simply due to a passive uptake of maternal immunoglobulins. The spleen precedes the bone marrow for a high level of expression of the paternal allotype. In the thymus, cells which contain cytoplasmic immunoglobulin are found at low frequency, but their absolute number represent a substantial contribution to the B lineage cell pool. A small proportion of such thymus cells do not show allelic exclusion of kappa chain allotypes.     

Role of IgD in the immune response and tolerance. II. Precursor analysis of murine B cells separated on the basis of surface IgD or treated with an anti-delta serum.

Precursor analysis of the function of murine B cells with surface IgD has been accomplished using an alloantiserum directed against a delta allotype and cellular affinity chromatography. Spleen cells from 3-week-old C57BL.Ige mice were treated with a TNP-labeled anti-delta allotype serum, washed and passed through an anti-TNP affinity column. The cells passing such a column were depleted of IgD-bearing lymphocytes by immunofluorescence and also showed a greatly reduced ability to develop into specific antibody-forming cell clones vs. the fluorescein (FL) hapten in a limiting dilution microculture system in vitro. In contrast, the IgD-bearing cells eluted from such columns were slightly enriched in precursor activity compared to the original population. Moreover, brief treatment with anti-delta serum alone produced a partial reduction in the precursor frequency vs. FL with C57BL.Ige spleen cells (which bear the appropriate delta allotype) but not C57BL/6 spleen cells (which are negative for this delta allotype). This effect varied with the age of the spleen donor, but was most dramatic with splenocytes from 2 to 3-week-old mice. This contrasts with the effect of anti-mu pretreatment, which only inhibited neonatal precursors. The results are interpreted in terms of the maturation of murine B cells and the function of IgD and IgM receptors in triggering an immune response.     

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.     

Allotypic determinants on the surface of rosette-forming cells in the rabbit

Antigen-binding cells to sheep erythrocytes from non-injected rabbits and from rabbits after a single injection of sheep red blood cells (SRBC) have been detected by the immunocyto-adherence technique (rosette formation). The expression of immunoglobulin allotypic determinants on these antigen-binding cells has been investigated by inhibition of rosette formation with anti-allotype antisera. The results indicate that a and b locus allotypes are expressed by the great majority of rosette-forming cells (RFC) in the lymph nodes of injected and non-injected rabbits. Ms3 allotype is expressed by practically all RFC from non-injected rabbits, while, after injection of SRBC, a population of RFC appears, which does not express this allotype. An antiserum to d 11 consistently failed to inhibit RFC. Pretreatment of lymphocytes from rabbits heterozygous at the b locus with antiserum to only one allelic product produced an inhibition of rosette formation which was approximately half that obtained when antiglobulin reagents to both allelic products were added. The results suggest that in heterozygous animals, the two allelic markers are expressed on two different populations of antigen-binding cells. Electron microscopy studies on the morphology of RFC showed that the majority of the RFC were formed around small lymphocytes.