Investigation of immunomodulatory properties of recombinant bovine complement component C3d
Immune responses of young calves are less vigorous than those of mature cattle. This results from both the immaturity of the neonate’s immune system and interference by passive maternal antibodies. Compared to adults, young animals have low concentrations of many factors that upregulate immune response including complement protein C3. In immune responses, a fragment of C3, C3d, binds to B lymphocytes and helps signal cellular proliferation and the subsequent production of antibodies. In previous OCA supported research we successfully isolated and characterized the gene coding for bovine C3d. We have now created a recombinant bovine C3d (rBovC3d) that permits investigation of its the biological properties, in particular its effects on neonatal B cells. Eventually it is hoped that inclusion of rBovC3d as an adjuvant will improve the efficacy of vaccines used to immunize neonates. One strategy would be incorporation of both C3d and antigens in ISCOMs (immune stimulating complexes), microparticles that have also been shown to help overcome the suppressive effects of maternal antibodies. We have conducted initial studies in 4 and 6 week old calves looking at the effects of ISCOM vaccines containing antigens of Mannheimia haemolytica, a contributor to enzootic pneumonia of calves. We have also shown that C3d can be incorporated into ISCOMs. The next logical step will be creation of ISCOM vaccines containing both M. haemolytica antigens and C3d. C3d exerts its effect by binding to specific receptors on B cells, identified as CD21. Therefore we have also compared the expression of CD21 on neonatal white cells to expression on similar cells obtained from mature cattle. Perhaps not surprisingly, we discovered that fewer neonatal cells express CD21, indicating that poor response is a consequence of both low levels of C3 and decreased recognition of C3d. This suggests that response may be further enhanced by inclusion of modulators which could up-regulate CD21 expression. While binding of C3d to B cells provides an up-regulatory stimulus, that effect can be countered by binding of maternal IgG antibodies to Fcg receptors (FcgRIIb2) on the same cells. Although FcgRIIb2 had been identified on bovine cells, prior to this research, there was no information available describing the concentration or distribution on neonatal lymphocytes. Therefore, we also initiated studies looking for FcgRIIb2 expression on white blood cells of neonates compared to adults. In order to do this we attempted to isolate the gene coding for bovine FcgRIIb2, and were surprised to find, not one but four genes coding for variations of this receptor: FcgRIIb2, reported previously, and FcgRIIb1, both similar to human receptors that are known to be down-regulatory; FcgRIId, not previously reported in cattle, but with a cytoplasmic motif similar to an up-regulatory receptor (CD16) in humans and mice; and a novel soluble form FcgRIIb2, which we suspect would contribute to interference with active immune response. This discovery greatly complicates the picture for B cell regulation in cattle; however, differential expression of these receptors may explain why the presence of maternal IgG in neonates can have greater inhibitory effects than observed in mature animals. Monoclonal antibodies to CD32, an alternative name for extracellular regions FcgRIIs, can detect expression on lymphocytes, but does not distinguish between the activating and inhibitory forms of the receptor. We have completed some studies demonstrating differences in the expression of CD32 on various white cell populations and in the concentration of CD32 on B cells of neonatal calves compared to adults, but will need to work further with the genes we have isolated to find ways to determine if receptors expressed on neonatal cells are primarily of the down regulatory type. This information will have great impact on vaccine design, since it could allow for incorporation of material that alters expression of FcgRIIs or the intracellular signalling following binding of maternal antibodies to FcgRIIs. Together improved understanding of the regulatory effects of C3d and FcgR’s on neonatal B cells will move the research much closer to the ultimate goal, efficacious vaccination of neonatal calves.
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