This is the first time the magnitude and the kinetics of the T-D and T-I2 antigen specific B cell response has been described in cattle. Briefly, the response to the T-D antigen was characterised by an expansion of TNP-specific plasma cells peaking at day 7 post-TNP primary immunisation, which was associated with a subsequent increase in the titre of TNP-specific IgG antibodies. Following booster immunisation, a larger TNP-specific plasma cell burst was detected at day 3 to 5 post-TNP boost, and was similarly associated with a larger increase in TNP-specific IgG titres. The timing and magnitude of the T-D antigen-specific plasma cell bursts is in agreement with the findings of a previously published study, in which calves were immunised with another T-D model antigen, ovalbumin. Moreover, a plasma cell burst has also been previously detected (with identical kinetics: 6 to 7 days post-primary immunisation) in humans following their immunisation with a number of different antigens, such as inactivated influenza virus, respiratory syncytial virus, serogroup C meningococcal polysaccharides and tetanus toxoid. The timing and magnitude of the secondary plasma cell burst and increased IgG response, i.e. a rapid and larger secondary response, is in keeping with the central dogma of immunological priming by a T-D antigen[15, 22].
There was an increase in the number of TNP-specific memory B cells from day 3 post-TNP-CGG boost that peaked at day 6 to 7 post-TNP boost. TNP-specific and CGG-specific memory B cells were still detectable in the blood of calves from the T-D group at day 34 post-TNP boost. The timing and magnitude of this T-D antigen specific response, following booster immunisation, is in keeping with other previously published data. Although no TNP-specific memory B cells were detected in the T-D group following the primary immunisation. Previous studies in both humans and cattle have shown low numbers of antigen-specific memory B cells following primary immunisation with a T-D antigen[4, 15]. The rapid induction of TNP-specific plasma cells from day 3 post-TNP boost and the associated increase in TNP-specific IgG antibody titres observed in the T-D group suggests that a number of TNP-specific memory B cells were indeed generated after primary immunisation, but the quantities in blood were below the detection levels of our ELISPOT assay.
T-independent antigens are unable to prime individuals for a secondary immune response. In this study the T-I2 group showed no detectable TNP-specific plasma or memory B cells, after either the primary or booster immunisation with TNP-AECM-FICOLL. Despite the lack of detectable antigen-specific plasma or memory B cells in the blood, there was a small increase in TNP-specific IgG titres in this group following primary and booster immunisations. It is likely that this increase in antibody titres results from the in situ generation of short-lived plasma cells, as previously described. These findings are in keeping with Fink et al. who suggest that the absence of specific T cell help results in reduced germinal centre formation, which promotes an extra follicular plasma cell response, favouring short-lived plasma cell formation. Kelly et al. have also shown that following primary immunisation with a T-I2 antigen, serogroup C meningococcal capsular polysaccharide, very low/undetectable numbers of antigen-specific memory B cells were generated. The results from the present study shows that in cattle the kinetics and magnitude of the T-I2 antigen-specific plasma and memory B cell response is similar to that of the human and mouse.
Immunisation with the third party antigen (TTC) in this study resulted in similar TTC-specific plasma and memory B cell kinetics that have been previously described for other TD antigens[4, 15, 20, 21]. Briefly, a burst of TTC-specific plasma cells was detected from day 7 to 14 post-TTC primary immunisation and from day 3 to 5 post-TTC boost in all animals. Interestingly, we found that the TTC-specific plasma cell bursts in the T-D group were also associated with a small increase in the number of TNP- and CGG-specific plasma cells post-TTC primary immunisation and/or boost. These results are consistent with those previously published by Bernasconi et al., which demonstrated a bystander polyclonal differentiation of memory B cells into plasma cells specific for various recall antigens (Toxoplasma gondii and measles virus) following the immunisation of humans with an unrelated T-D antigen (tetanus toxoid).
There was a steady increase in the number of TTC-specific memory B cells post-TTC primary immunisation followed by at least a 14 fold increase in the number of these cells post-TTC boost. The T-D group also showed an increase in the number of TNP- and CGG-specific memory B cells only post-TTC boost (at day 6–8 post-TTC boost). Based on the timing of emergence of the TNP- and CGG memory B cells, it is unlikely that these memory B cells were generated from newly activated naïve B cells, but instead suggests that pre-existing TNP- and CGG-specific memory B cells were subjected to one (or more) round(s) of antigen-independent (bystander) proliferation within the T-cell regions of the draining lymph node. This bystander stimulation of T-D antigen-specific plasma and memory B cells may provide a mechanism for maintaining long term T-D antigen-specific antibody titres.
In conclusion, the present study showed that bystander stimulation of an established T-D B cell memory response may occur in cattle, providing a possible mechanism of maintaining protective antibody titres for long periods of time. Our findings also provided a better characterisation of the key differences between T-D and T-I immune responses in cattle. However, as highlighted by Fink et al., more studies are required to further elucidate the mechanisms of the T-D and T-I2 activation of antigen-specific plasma cells. As demonstrated by this study and others[4, 15, 22], clearly the timing and magnitude of the antigen-specific plasma cell response is dependent on a number of factors, including the nature of the immunising antigen.
This knowledge will be particularly useful in elucidating the B cell response to the largely T-I antigen, FMDV. Indeed as mentioned previously, most natural pathogens contain both T-I and T-D antigens, but viruses such as FMDV tend to preferentially generate a T-I immunological response. FMDV is a highly contagious virus infecting cloven-hoofed animals, leading to skin erosions of the cutaneous mucosa. The virus has a significant global socio-economic impact, and the maintenance of FMDV-free status is critical for the free trade of animals and animal products. One of the main methods of FMDV disease control and eradication is through vaccination. The current FMDV vaccine only promotes short-term humoral immunity (presumably due to the essentially T-I nature of this virus), and regular repeat vaccinations are used to maintain protective IgG antibody titres, due to the lack of long-term immunological protection. Therefore, future studies including a more detailed assessment of the B cell response against FMDV in cattle could provide important data to underpin a better evaluation of novel candidate vaccines that induces a robust and sustained T-D rather than a T-I response.