Immunohistochemical localization of feline immunodeficiency virus using native species antibodies

Abstract

Feline immunodeficiency virus (FIV) is the feline analog of human immunodeficiency virus and a small animal model of human acquired immune deficiency syndrome (AIDS). We sought to identify early in vivo target cells in cats infected with clade B or C FIV. In tissues, however, neither mouse monoclonal nor rabbit polyclonal antibodies suitably detected FIV because of either insensitivity or lack of specificity. We therefore developed an immunohistochemical protocol using high-antibody-titer serum from cats chronically infected with FIV(Petaluma). Native species anti-FIV antibodies were labeled with biotinylated protein A before placement on tissues, and downstream signal was tyramide-amplified. This method revealed many productively infected cells in bone marrow, lymph node, thymus, mucosal-associated lymphoid tissue, and spleen, but few such cells in liver and none in kidney or brain. Concurrent labeling for virus and cell phenotype revealed that antigen-bearing populations were primarily T lymphocytes but included macrophages and dendritic cells. Our results demonstrate that FIV: 1) expands rapidly in T cells, 2) targets long-lived reservoir populations, and 3) is replicatively quiescent in brain at 3 weeks after infection. Use of native species antibodies for immunohistochemical detection of infectious antigens has application to other settings in which xenotypic (eg, mouse and rabbit) antibody sources are inadequate or unavailable.

Figure 1.

Mean FIV blood mononuclear cell supernatant tissue culture infectious doses (TCID/ml) and plasma viral RNA loads (copies/ml) 3 weeks after inoculation in cats infected with FIV-B-2542 or FIV-C-Pgmr.

Figure 2.

FIV chromogenic immunohistochemistry. a: FIV antigen⁺ cells in lymph node germinal center (large arrow) and paracortical cells (small arrow). b: No FIV antigens in sham-inoculated control cat lymph node. c: Scattered labeled cells (arrows) by RNA in situ hybridization in lymph node from FIV⁺ cat. FIV immunohistochemistry on tissues from FIV⁺ cat including: thymic lobule with clustering of antigen⁺ cells along junction between cortex (C) and medulla (M), with additional antigen localization in or near Hassal’s corpuscle (HC) (d, arrow); viral antigen expression in splenic periarteriolar lymphatic sheaths (e); abundant FIV antigen in ileal lamina propria leukocytes and mucosal-associated lymphoid tissue (Peyer’s patch) germinal centers (f); one antigen⁺ mononuclear cell (arrow) in lumen or lining hepatic sinusoid (g); apparently cell-free virus lining endothelial surface of small venule in kidney (arrow), but not in renal tubules or larger artery (at left; h); and brain with no detectable viral antigens (i). Original magnifications: ×200 (a, c, and e), ×100 (b and d), ×240 (f), ×600 (g), ×400 (h), and ×40 (i).

Figure 3.

Fluorescence immunohistochemistry. a: Diagram of three-color immunofluorescence assay demonstrating prelabeling of heterologous antiserum with biotin-protein A followed by tissue binding and Cy3-tyramide amplification for red labeling of FIV, fluorescein isothiocyanate-conjugated secondary antibodies for green labeling of cell phenotype antibodies, and DAPI counterstaining for blue labeling of nuclear chromatin. b: FIV antigens co-localized with CD3⁺ T cells in lymph node paracortex. c: An FIV⁺ AM-3K-labeled macrophage (yellow arrow) and a macrophage without FIV antigens (green arrow) in lymph node medulla. d: S-100⁺ dendritic cells with (yellow arrow) and without (green arrow) FIV antigens in lymph node follicle; also present is a FIV⁺ S-100⁻ cell (red arrow). e: A few cytokeratin⁺ thymic epithelial cells (yellow arrows) as well as cytokeratin⁻ FIV⁺ cells (red arrow) that are probably mature thymocytes in medulla. f: FIV antigens in both CD3⁺ (yellow arrow) and CD3⁻ (red arrow) cortical thymocytes. g: CD45R/B220⁺ B cell infiltrates, occasionally forming pseudofollicles (outlined) in thymus of FIV-infected cat. Original magnifications: ×1000 (b and d), ×600 (c and f), ×400 (f), ×200 (e), and ×100 (g).