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Limited active antibody responses in neonates following vaccination have been attributed to immaturity of the immune system and to the suppressive effects of maternal antibodies. The activating receptor CD21 (CR2), when co-ligated with membrane IgM (mIgM) by complement-bound antigen lowers the threshold for activation of B lymphocytes. The inhibitory receptor CD32 (FcgammaRII) when co-ligated with mIgM by antigen-antibody complexes raises the threshold for activation. Expression of these receptors, which potentially play roles in regulation of B cell responses in the presence of maternal antibodies in neonates, has been recently characterized in blood lymphocytes in neonatal calves. Little is known however about expression of these receptors in the lymphoid tissues, where immune responses are initiated. In this study, expression of CD21, mIgM and CD32 receptors by B lymphocytes was studied in a range of lymphoid tissues including spleen, lymph nodes and bone marrow from newborn and 7-week-old calves using flow cytometry. The proportion of naïve B lymphocytes in the lymphocyte gate was significantly lower in blood and spleen of newborn calves compared to 7-week-old calves. Over 90% of B lymphocytes expressed CD21 in the lymphoid tissues. In the lymph nodes and spleen, a lower proportion of mIgM(+) B lymphocytes expressed CD32 compared to blood. In addition, intensity of expression of CD32 on B cells in lymph nodes was significantly lower compared to that in blood, suggesting a lower potential for inhibitory signalling in B cells in the lymphoid microenvironment. Investigation of the CD5(+) B cell population (as an indicator of B1 B cells) suggested an increase in the proportion of IgM(+)CD5(+) cells with age in calves, in both blood and lymphoid tissue, in contrast to the situation in humans and mice. Overall, the majority of naïve B lymphocytes in lymphoid tissues in neonatal calves expressed both activating (CD21, mIgM) and inhibitory (CD32) receptors. These receptors may provide targets for novel adjuvants, to lower the threshold for activation of B cells in neonates, and enhance antibody responses., (Copyright 2010 Elsevier B.V. All rights reserved.)

Receptors for the Fc portion of immunoglobulin molecules (FcR) provide an important and vital link between circulating antibody and cellular effector functions. These receptors have been well characterized in human and murine species, however few of these receptors have been investigated in livestock. FcgammaRII (CD32) is an FcR previously shown in mice and humans to exist in multiple isoforms, both activating (FcgammaRIIa, FcgammaRIIc) and inhibitory (FcgammaRIIb), on a wide variety of cells including B cells, T cells, dendritic cells, monocytes, macrophages and platelets. On B cells, FcgammaRIIb acts to suppress cell activation and immunoglobulin production by means of an intracellular immunoreceptor tyrosine-based inhibitory motif signaling domain. Two sub-isoforms of FcgammaRIIb, designated b1 and b2, distinguished by the inclusion of an additional cytoplasmic exon in the b1 form, have been demonstrated in humans and mice, whereas only one sequence corresponding to the human and mouse b2 isoform has been identified in cattle. In this study, the expression profile of FcgammaRIIb in bovine blood mononuclear cells was characterized by collecting blood samples from mature cattle of dairy and beef breeds, and determining their FcgammaRIIb mRNA expression profile by RT-PCR. Analysis revealed the presence of two uncharacterized bovine FcgammaRIIb transcripts in addition to the single previously published transcript. Analysis of the first unknown transcript revealed high homology with published human and murine FcgammaRIIb1 sequences. This transcript was present in all cell types examined, with little variation in primary sequence between individuals or among breeds. The second unknown sequence was found to be homologous to the murine FcgammaRIIb3 (IgG-binding protein or soluble FcgammaR in humans) sequence. This transcript appears to have a much more limited expression profile, which may indicate that expression varies with the cellular activation-state of the cell. These results indicate that cattle, like humans and mice, express multiple sub-isoforms of FcgammaRIIb. These findings add further complexity to the regulation of IgG-mediated immunity and provide new insight into the role Fc receptors play in antigen acquisition and presentation in cattle., (Copyright 2009 Elsevier B.V. All rights reserved.)

It is difficult to induce active immune responses in neonatal calves, partly due to limited functional ability of the immune system and partly due to immune inhibitory effects of maternal antibodies. CD21 (complement receptor 2), an activating receptor, and CD32 (Fc gamma receptor II), an inhibitory receptor, can both be expressed by mature B lymphocytes. Studies of the signalling pathways regulating B cell activation suggest that these receptors have mutually antagonistic effects, that may determine immune responsiveness in the first months of life. In a cross-sectional study, blood was collected from 41 Holstein calves, 1-90 days of age, and 12 mature cows. The absolute number of CD21 and CD32 positive cells increased from birth until 90 days of age, with CD21+ cells showing a greater relative increase compared to CD32+ cells. Approximately 89% of CD21+ cells also expressed CD32. In calves, CD32+ cells consisted of two distinct populations, characterized to be CD14+CD32+IgM(-) (44%) and CD14(-)CD32+IgM+ (53%) cells, consistent with monocytes and B cells respectively. Mean fluorescence intensity (MFI) for CD21 did not change appreciably up to 90 days of age, and mean values were slightly lower than for adults. MFI for CD32 on CD21+ lymphocytes increased slightly over the first 3 months of life, but values were lower than for adults. Over 92% of calf membrane immunoglobulin M+ (mIgM) B cells expressed CD21, however only 60-80% of CD21+ cells were mIgM positive, regardless of age. Although the CD21+IgM(-) cells were not characterized further, it was evident that CD21 is not a suitable surrogate marker for B lymphocytes in calves. Most importantly this study showed that from birth, the majority of circulating B cells express both CD21 and CD32. This suggests that these cells are subject to activating and inhibiting influences mediated by these receptors from birth. Expression of CD21 does not appear to be a limiting factor in neonatal B cell responses.

The gene fragment coding for bovine C3d gene (boC3d) was cloned and expressed as a component of fusion proteins destined for use in vaccine studies in cattle, and for in vitro experiments. This fragment of complement protein C3 (C3d) has been shown to enhance B cell responses when complexed with antigen. Three potential vaccine constructs were engineered to contain one, two or three boC3d units linked to a fragment of the leukotoxin of Mannheimia haemolytica A1, an economically important pathogen of cattle that causes a fibrinous pneumonia in calves. A recombinant biotinylated boC3d protein (for use in in vitro studies) was generated by endogenous biotinylation in Escherichia coli by means of the BirA holoenzyme synthetase. All recombinant proteins incorporated polyhistidine tags and were purified by nickel-agarose chromatography, then analyzed by SDS-PAGE and Western immunoblot. The identity of boC3d was confirmed by mass spectrometry, since monoclonal antibodies to boC3d were not available. To date, published research into the adjuvant activities of C3d has been limited to experiments in mice and rabbits, using antigens unrelated to diseases occurring naturally in these species. The boC3d fusion proteins expressed in this study will provide the basis for immunization trials in cattle and studies of receptor binding and cell activation of bovine lymphocytes.