Impaired lymphocyte functions in heterozygous rabbits recovering from neonatal allotype suppression

Lymphocytes from heterozygous rabbits suppressed for an allotypic determinant on kappa light chains by exposure to maternally derived antibodies specific for the paternal gene product were analyzed for their capacity to express membrane-bound and secreted immunoglobulin (Ig). Individual cells displaying allotypic membrane Ig (mIg) were enumerated by a rosette test, while Ig-secreting cells were assessed by means of a hemolytic plaque assay. In a group of suppressed rabbits varying in age from 3 to 19 months, the proportion of cells with mIg of the paternal type was markedly higher than that of cells secreting that type of Ig. The same high proportion of lymphocytes displaying mIg of the suppressed type was observed whether lymphocytes from blood, spleen, or lymph nodes of suppressed rabbits were examined. In contrast, similar analyses performed with cells of normal heterozygous rabbits showed no discrepancy between mIg expression and secretion of either allotype. Lymphocytes synthesizing Ig of the paternal type were also defective in responses to lipopolysaccharide (LPS), which stimulates differentiation to Ig secretion in normal B lymphocytes. These results support the idea that B lymphocytes capable of synthesizing the suppressed type of Ig have functional impairments affecting secretion and responses to environmental stimuli.     

Precocious recovery from allotype suppression in transiently chimeric rabbits

Rabbits suppressed for paternal immunoglobulin allotypes specified by the b locus were injected with spleen cells from non-inbred donors at the age of 1 month. This resulted in transient chimerism as shown by the appearance of 10 to >200 μg of donor type Ig per milliliter recipient's serum 1 to 2 weeks after cell transfer. Antibody production by donor cells could not be demonstrated during their survival in the recipients. If, and only if, the donor cells produced Ig of the suppressed allotype, the release from suppression was expedited as judged by the time of appearance and increase of lymphoid cells with membrane-bound Ig of the suppressed type, and also by the onset and rise of secreted Ig of this type in the recipients' sera.     

In vitro allotype suppression of spleen cells from immunized rabbits

We tested whether rabbit immune lymphocytes could be suppressed by anti-allotype antibody (Ab) in vitro as shown for normal lymphocytes. Spleen cells (SpC) from rabbits heterozygous at the b locus (b⁴b⁵) of immunoglobulin (Ig) κ chains were treated with IgG preparations of anti-b4 or anti-b5 Ab in vitro for 24 hr (day 1). After this treatment, the SpC were washed and recultured in medium to day 5. The secreted b4- and b5-Ig were quantitated by a radioimmunoassay. SpC from rabbits injected once with sheep red blood cells (SRBC) were allotype-suppressed. Thus, these SpC treated with anti-b4 Ab secreted normal amounts of b5-Ig but secreted much lower amounts of b4-Ig. Similarly, SpC treated with anti-b5 Ab secreted normal amounts of b4-Ig but secreted no detectable b5-Ig. In contrast, SpC from rabbits injected several times with SRBC (hyperimmunized) could not be allotype-suppressed. Hence, the susceptibility of primary immune cells and the resistance of hyperimmune cells to suppression appear to depend on the stage of B-lymphocyte differentiation, presumably because of loss of surface Ig or perhaps because of other changes in the cells as they differentiate during the immune response.     

In vitro immunoglobulin allotype synthesis by spleen cells of heterozygous rabbits. Specific suppression by anti-allotype antibody

The production of immunoglobulin (Ig) bearing the b4 and b5 allotypic markers by b⁴b⁵ heterozygous spleen cells cultured in vitro was assessed by means of a sensitive and reproducible radioimmunoassay. Ig synthesis was demonstrated by the increasing amounts of the b4 and b5 allotypes appearing with time in the supernatant fluids. To determine the effect of anti-b4 or anti-b5 antibody on the synthesis of the b4 and b5 allotypes, spleen cells from b⁴b⁵ heterozygous rabbits were incubated for 24 hr in the presence of anti-b4 or anti-b5 and then washed and cultured for an additional 4 days. Anti-b4 suppressed the production of the b4 allotype with no effect on b5 production, whereas anti-b5 suppressed the production of b5 allotype with no effect on b4 production. This suppression of allotype synthesis in vitro presumably results from an antigen-antibody reaction occurring on the surface of lymphoid cells by a mechanism which may be similar to that which brings about allotype suppression in vivo for fetal and newborn rabbits.     

Partial amino acid sequence of a rabbit immunoglobulin light chain of allotype b5

The amino acid sequence of a rabbit immunoglobulin light chain of allotype b5 has been nearly completed. A comparison of its structure with that of light chains of allotypes b4, b6, and b9 confirms that the constant regions of these various kappa chains differ by 20-35%. The substitutions are clustered in parts of the second half of the chain, and the b5 form bears more resemblance to the b6 chain than to any other, in good agreement with previous serological data. The analysis of the variable region reveals the existence of certain allotype-associated residues which have also been reported in other b5 chains, but not in proteins of the other allotypes. An examination of the rabbit light chain sequences between positions 96 and 107 suggests that this portion of the chain may be encoded separately by a joining "J" DNA segment, as has been described previously for murine and human immunoglobulins. In the rabbit, however, these J kappa regions appear to differ from one allotype to another. Together with the extensive variations of the constant regions, these data suggest that the rabbit kappa gene organization more closely resembles the murine gamma system (four different C gamma genes each flanked by its J segment) than the murine kappa system (a single C kappa gene).     

Selective suppression of allotype expression induced in vitro: maintenance of suppression following adoptive transfer

The question of the ultimate fate of lymphocytes subjected to treatment with anti-allotype antibody (Ab) has been investigated by means of an adoptive transfer system that uses noninbred rabbits matched for major histocompatibility antigens and mismatched for allotype. Suppression of b4 immunoglobulin (Ig) production was induced by incubating lymphocytes from b4b5 rabbits with anti-b4 in culture. Transfer of b4-suppressed cells to newborn recipients of allotype b6b6 resulted in stable chimerism of mixed donor-recipient allotypes, in which b4 Ig production remained suppressed. In recipients of non-Ab-treated cells, b4 Ig production predominated over b5, as had been the case in the intact donor. No evidence for stimulation of b4 Ig synthesis was seen, even when lymphocytes and serum from 1-week-old recipients were examined. When lymphoid cells of antigen-primed b4b5 donors were treated with anti-b4 in vitro and transferred, Ab production of the b4 type was specifically suppressed, with compensatory over-production by Ab-forming cells of the b5 type. The results reported here indicate that although anti-allotype Ab is not directly cytotoxic, a significant proportion of the b4-committed cells were irreversibly inactivated as a result of Ab pulse treatment.     

Production of allotype by bone marrow cells transferred to rabbits subjected to suppressor treatment by allotype

Inbred rabbits of B/J strain were immunized against Salmonella typhi and their bone marrow cells plus LPS were injected into (Chbb: HM X B/J) F1 hybrids at the age of 3-7 weeks, which had received at birth a treatment of the allotype suppression. Antityphoid antibodies bearing the suppressed allotype were found in the serum of the recipients, which showed no signs of the graft versus host reaction.     

Defective granule exocytosis in Rab27a-deficient lymphocytes from Ashen mice

Because mutations in Rab27a have been linked to immune defects in humans, we have examined cytotoxic lymphocyte function in ashen mice, which contain a splicing mutation in Rab27a. Ashen cytotoxic T lymphocytes (CTLs) showed a >90% reduction in lytic activity on Fas-negative target cells compared with control C3H CTLs, and ashen natural killer cell activity was likewise diminished. Although their granule-mediated cytotoxicity pathway is profoundly defective, ashen CTLs displayed a normal FasL-Fas cytotoxicity pathway. The CD4/8 phenotype of ashen T cells and their proliferative responses were similar to controls. Ashen CTLs had normal levels of perforin and granzymes A and B and normal-appearing perforin-positive granules, which polarized upon interaction of the CTLs with anti-CD3-coated beads. However, rapid anti-CD3-induced granule secretion was drastically defective in both CD8(+) and CD4(+) T cells from ashen mice. This defect in exocytosis was not observed in the constitutive pathway, as T cell receptor-stimulated interferon-gamma secretion was normal. Based on these results and our demonstration that Rab27a colocalizes with granzyme B-positive granules and is undetectable in ashen CTLs, we conclude that Rab27a is required for a late step in granule exocytosis, compatible with current models of Rab protein function in vesicle docking and fusion.     

Modulation and regrowth of allotype on normal rabbit peripheral blood lymphocytes: allelic inclusion of b4 and b6 in single cells.

Allelic inclusion at the b-locus by heterozygous peripheral blood rabbit lymphocytes was demonstrated by using the mixed antiglobulin techniques (6, 7, 19). Heterozygous cells (64, 6) were treated with monospecific antiallotype reagents at 4 °C and warmed at 37 °C. The removal of surface allotype determinants was studied and, both the b4-and b6-specificities co-modulated after sensitization with either anti-b4 or anti-b6. Experiments were undertaken in which cells stripped of one or both allotypes by antiallotype induced modulation were cultured overnight. Allotype was then regenerated. Double expression of allotype by cells before antiallotype treatment was recovered following overnight regrowth at levels equal to those seen before treatment. Such was not the case when b44 and b66 cells were cultured together. These results indicate that normal heterozygous peripheral blood lymphocytes may express both b-locus alleles and that these determinants are in some manner physically associated with one another in the cell membrane.     

Defective control of cytoplasmic calcium concentration in T lymphocytes from old mice

Cytoplasmic calcium concentration ([Ca]i) rises within minutes of exposure of T lymphocytes to a mitogen. T cells from old mice are defective in this reaction, a defect that could reflect either altered signal transduction or instead a more general age-associated change in intracellular calcium regulation. We therefore tested the ability of T cells from old mice to regulate their [Ca]i concentration after exposure to low concentrations of ionomycin, an agent that raises [Ca]i but bypasses receptor-mediated signal transduction mechanisms. Exposure of T cells to ionomycin leads to an abrupt increase in [Ca]i followed by stabilization at a dose-dependent plateau level that is affected by extracellular EGTA, by calmodulin inhibitors, and by modulators of protein kinase C. Plateau levels of [Ca]i after ionomycin challenge were consistently lower in T cells from old mice than in T cells from young mice. Flow cytometric experiments showed that while essentially all T cells from both old and young mice responded to ionomycin, they did so to an extent that depended on donor age. The age-dependent increase in resistance to ionomycin-induced changes in [Ca]i cannot be attributed to diminished membrane permeability to the ionomycin-calcium complex. The data suggest that aging may lead, in T lymphocytes, to a relative resistance to increases in [Ca]i, a resistance that in turn prevents cell activation.     

Variation in the N-terminal sequence of heavy chains of immunoglobulin G from rabbits of different allotype

The sequences of the N-terminal peptides prepared by Pronase digestion of the heavy chain of rabbit immunoglobulin G of allotype Aa1, Aa2 and Aa3 were determined and were shown to be related to the allotype. An N-terminal fragment of about 34 residues was also prepared from the allotype heavy chains, by cleavage with cyanogen bromide; the yield varied with the allotype. The sequences of the cyanogen bromide fragments from the Aa1 and Aa3 heavy chains contain allotype-related variations similar to those found in the N-terminal Pronase peptides, and these sequences are thought to be representative of the whole heavy-chain populations. There is about 60% homology between the two sequences, and superimposed on the differences between them there are a number of positions within each sequence at which at least two amino acids are present.     

Evidence for active synthesis of two allelic b locus markers by single lymphocytes from heterozygous rabbits.

The double expression on single cells of both b4 and b6 allotypic markers by the mixed antiglobulin technique was observed on single peripheral blood lymphocytes (PBL) from heterozygous rabbits which were treated with pronase and incubated overnight to allow for regrowth of surface immunoglobulin (Ig). The percent of double staining rosettes observed after lymphocyte processing prior to pronase stripping ranged from 10–30%. Following pronase treatment at concentrations allowing for maximum stripping but assuring high cellular viability, the total rosetting population was reduced to 1–3% in the majority of cases. Occasionally, pronase stripping was less effective. Overnight incubation in serum free medium resulted in the reappearance of surface Ig with double staining rosettes ranging from 8–30%. Exposure of b6 or b4 normal rabbit serum to b4 or b6 cells, respectively, resulted in uptake of b6 in which most of the passively acquired Ig eluted off during overnight incubation, while negligible uptake of b4 was seen. Coincubation of normal b4, 4 PBL which presumably are capable of donating Ig with pronase treated b6, 6 PBL and vice versa also resulted in the absence of mixed rosettes. That simultaneous active synthesis of both allelic markers results in double allotype expression was demonstrated by kinetics data. Supporting this was the observation that only after washing of cycloheximide inhibited cells did allotype reexpression resume.