Optimized Multicolour Immunofluorescence Panel for Cattle B Cell Phenotyping by an 8-Colour, 10-Parameter Panel

This 8-colour, 10-parameter panel has been optimised to distinguish between functionally distinct subsets of cattle B cells in both fresh and cryopreserved peripheral blood mononuclear cells (PBMCs). Existing characterised antibodies against cell surface molecules (immunoglobulin light chain (S-Ig(L)), CD20, CD21, CD40, CD71 and CD138) enabled the discrimination of 24 unique populations within the B cell population. This allows the identification of five putative functionally distinct B cell subsets critical to infection and vaccination responses; 1) naïve B cells (BNaïve), 2) regulatory B cells (BReg), 3) memory B cells (BMem), 4) plasmablasts (PB) and 5) plasma cells (PC). Although CD3 and CD8α can be included as an additional dump channel, it does not significantly improve the panel’s ability to separate “Classical” B cells. This panel will promote better characterisation and tracking of B cell responses in cattle as well as other bovid species as the reagents are likely to cross react.


Background:
As our knowledge of immune cell subsets and their functions increases, so does the need to identify and measure alterations in their phenotype and frequency. The mammalian B cell population consists of several functionally distinct subsets that together comprise the major mediator of humeral immunity (1,2) . The development of naïve B cells (BNaïve) is important for long term immune protection (3)(4)(5). Driving the development of antibody secreting cells (ASC) and memory B cells (BMem) is an essential requirement of many vaccines that elicit neutralizing antibody responses (6)(7)(8). Furthermore, these subsets are often the source of therapeutic antibody candidates (as vaccines or immunotherapies) against infectious diseases (6)(7)(8).
Regulatory B cells (BReg) also play a vital role in suppressing infectious diseases (9,10). Consequently, the identification and relative quantification of B cell subsets is The capability to dissect B cell responses at high resolution is limited in many nonmodel species through a combination of limited reagents, lack of knowledge of species-specific B cell markers and standardised methods (11). This is certainly the case for cattle, a key food producing species and crucial for human nutrition globally, as a universal B cell lineage marker (i.e. CD19) and reagents against other well-known B cell subsets (e.g. IgD and CD38) are lacking (12). As technologies to design and deliver protective immunogens continue to emerge rapidly, it is essential to evaluate their applicability in other species as part of one health approaches.
Consequently, a need to study cattle B cell responses and their maturation at a high resolution.
Based on well characterised human and mouse B cell populations we hypothesise that these markers will identify five major subsets of B cells in cattle lymphocytes (Online Table 3): BNaïve, BMem, BReg, plasmablasts (PB) and plasma cells (PC) (13). The panel further allows for more in-depth characterisation of cattle B cells into 24 phenotypically unique subsets, following the gating strategy set out in Fig. 1; however, the functional discrimination and therefore importance between these subsets remains to be determined.
Our gating strategy consists of plotting CD40 against CD14 to select "classical" (CD40 + CD14 -) B cells. Although CD3 and CD8α are often used as a dump channel to isolate cattle B cells, their inclusion did not significantly improve separation (Online  (Fig. 1 A). Next, each population was further sub-divided by comparing CD71 against CD20 and sub-gated into CD71 + CD20 -SP, CD71 + CD20 + DP and CD71populations (Fig.1 B). Lastly, each of these sub-gates were divided as B Cell phenotyping in Cattle Roos et al. 5 either CD138 + or CD138 - (Fig.1 C), resulting in 24 minor subsets of cattle B cells. An important step while labelling the PBMCs is to first stain the cells with the CD20 antibody before adding any of the other antibodies in the panel (Online Figure 9).
Although this panel was developed and optimised on a BD LSRFortessa, it performed equally well using a BD Aria IIIU when sorting cattle B cell subsets for further molecular investigation. The panel also has potential to be adapted by moving the CD14 antibody into the Live/Dead channel (for example coupled to APC-Cy7), provided the monocytes do not need to be specifically analysed, or into an available violet channel, which will free the PE channel for an additional marker if needed (e.g. intra-cellular staining). The CD71 antibody in APC can also be moved into an available violet channel. Further potential to expand and tailor this panel includes the addition of a cattle cross-reactive human CD27 antibody, which could resolve the limitations in identifying BMem cells or the addition of an antibody against CD5, which is known to identify B1 cells in mice (22).  (11,12). Additionally, the panel allows the enrichment or B Cell phenotyping in Cattle Roos et al. 6 isolation of specific single B cells or their populations to further study function, specificity, and drive antibody discovery.

Similarity to published OMIPs:
None to date.

Acknowledgements:
The authors wish to acknowledge the valuable input of Dr. Kelcey Dinkel and Dr.