OMIP‐085: Cattle B‐cell phenotyping by an 8‐color panel

Abstract This 8‐color panel has been optimized to distinguish between functionally distinct subsets of cattle B cells in both fresh and cryopreserved peripheral blood mononuclear cells (PBMCs). Existing characterized 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 characterization 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 (B Naïve ) is important for long-term immune protection [3][4][5]. Driving the development of antibody secreting cells (ASC) and memory B cells (B Mem ) 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 (B Reg ) also play a vital role in suppressing infectious diseases [9,10]. Consequently, the identification and relative quantification of B-cell subsets is a fundamental requirement when evaluating pathogen or vaccine-induced immune responses and ultimately the development of better strategies to control diseases [1].
The capability to dissect B-cell responses at high resolution is limited in many non-model species through a combination of limited reagents, the lack of knowledge of species-specific B-cell markers and standardized methods [11,12]. 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 [13].
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, there is a need to study cattle B-cell responses and their maturation at a high resolution.
We have developed a flow cytometry panel using the existing antibodies against cell surface markers based on knowledge in humans and mice [14] (Table 1). With no pan B-cell markers known in cattle, such as CD19, CD72, or CD79α, we resolved B cells from other lymphocytes using CD14 (CCG33, [15]) to exclude the monocytes, CD40 (IL-A158, [16]) as a B-cell lineage marker, and included previously described cattle B-cell markers such as CD21 (CC21, [17,18]) and surface immunoglobulin light-chain (S-Ig(L), IL-A58, [17,19]) [2,20] ( Table 2). Subsets within these populations were further identified using the activation and differentiation markers CD71 (IL-A165, [21]), Based on well-characterized human and mouse B-cell populations, we hypothesize that these markers will identify five major subsets of B cells in cattle lymphocytes (Online Table 3): B Naïve , B Mem , B Reg , PB, and PC [14]. The panel further allows for more in-depth characterization of cattle B cells into 24 phenotypically unique subsets, following the gating strategy set out in Figure 1; however, the functional discrimination and therefore importance between these subsets remains to be determined (Tables 1 and 2).