Detection of Signal Regulatory Protein α in Saimiri sciureus (Squirrel Monkey) by Anti-Human Monoclonal Antibody

Abstract

Non-human primates (NHP) are suitable models for studying different aspects of the human system, including pathogenesis and protective immunity to many diseases. However, the lack of specific immunological reagents for neo-tropical monkeys, such as Saimiri sciureus, is still a major factor limiting studies in these models. An alternative strategy to circumvent this obstacle has been the selection of immunological reagents directed to humans, which present cross-reactivity with NHP molecules. In this context and considering the key role of inhibitory immunoreceptors-such as the signal regulatory protein α (SIRPα)-in the regulation of immune responses, in the present study, we attempted to evaluate the ability of anti-human SIRPα monoclonal antibodies to recognize SIRPα in antigen-presenting S. sciureus peripheral blood mononuclear cells (PBMC). As shown by flow cytometry analysis, the profile of anti-SIRPα staining as well as the levels of SIRPα-positive cells in PBMC from S. sciureus were similar to those observed in human PBMC. Furthermore, using anti-SIRPα monoclonal antibody, it was possible to detect a decrease of the SIRPα levels on surface of S. sciureus cells after in vitro stimulation with lipopolysaccharides. Finally, using computed-based analysis, we observed a high degree of conservation of SIRPα across six species of primates and the presence of shared epitopes in the extracellular domain between humans and Saimiri genus that could be targeted by antibodies. In conclusion, we have identified a commercially available anti-human monoclonal antibody that is able to detect SIRPα of S. sciureus monkeys and that, therefore, can facilitate the study of the immunomodulatory role of SIRPα when S. sciureus is used as a model.

Keywords: Saimiri sciureus; flow cytometry; immune response; non-human primates; signal regulatory protein α.

Figure 1

Flow cytometry analysis of anti-human signal regulatory protein α (SIRPα) monoclonal antibody cross-reactivity with Saimiri sciureus cells. Peripheral blood mononuclear cells (PBMC) were isolated from human or S. sciureus whole blood, stained with anti-SIRPα monoclonal antibody or isotype control and, then, analyzed by flow cytometry. Reactivity of anti-SIRPα antibodies [allophycocyanin (APC)] with human (A) and S. sciureus (B) PBMC was evaluated gating lymphocytes (P1) or monocytes populations, as defined by forward scatter (FSC) and sideward scatter (SSC) parameters.

Figure 2

Frequency of signal regulatory protein α (SIRPα)-positive cells in peripheral blood mononuclear cells (PBMC) samples from humans and Saimiri sciureus monkeys. PBMC were isolated from whole blood, stained with anti-human SIRPα monoclonal antibody and, then, analyzed by flow cytometry. Cells presenting SIRPα were quantified considering three main cells populations by morphological criteria: total PBMC, monocytes, and lymphocytes, as shown in Figure 1. Data represent mean ± SEM for five humans and seven monkeys.

Figure 3

Homology analysis of signal regulatory protein α (SIRPα) across primates. (A) Circular alignment of amino acid sequences of SIRPα protein in human and five non-human primates (Pan troglodytes, Gorilla gorilla, Macaca fascicularis, Callithrix jacchus, Saimiri boliviensis). The outer circle shows the amino acid scale. Green and gray bars on the second circle show the percent matching among all sequences used in the analysis. Inner circles show the sequence alignment in which each amino acid was represented by a different color. (B) Pairwise distance among all primates studied and (C) phylogenetic tree based on SIRPα protein alignments.

Figure 4

Prediction of linear B-cell epitopes in extracellular domain of signal regulatory protein α protein in Saimiri (A) and Homo sapiens (B). Linear B-cell epitopes were predicted to be located at the residues with the scores above 0.35 (yellow) and regions not predicted to be B-cell epitopes are under the threshold (green). The epitope score represents the average of the scores of least nine consecutive amino acids above the cut-off, and the sequences with higher mean values were detected as potential linear epitopes.

Figure 5

Modulation of signal regulatory protein α (SIRPα) levels by lipopolysaccharides (LPS) in Saimiri sciureus cells. Peripheral blood mononuclear cells (PBMC) of S. sciureus monkeys (n = 7) were incubated for 24 h in presence or absence (control) of LPS and, then, SIRPα was detected in monocytes population by flow cytometry using anti-human monoclonal antibody. (A) Frequency of SIRPα-positive cells in PBMC and (B) levels of SIRPα present on cell surface of SIRPα-positive cells (monocytes), as measured by mean fluorescence intensity. Data (mean ± SEM) are representative of two separate experiments. Statistical difference was tested by paired t-test in GraphPad Prism 5.0 software and p < 0.05 was considered significant.