TABLE 2.

RRV isolation and flow cytometric analysis of lymphocyte subpopulations obtained by immunomagnetic bead depletion^a

Rhesus monkey no.	Fraction type	RRV serology	Positive cells (%)b			Virus isolationc
			CD4+	CD8+	CD20+
1	Total PBMCs	Seronegative	51	35	8.4	−
	T-cell enriched		56	33	0	−
	B-lymphocyte enriched		0	19	69	−
2	Total PBMCs	Seropositive	38	37	12	−
	T-cell enrichedd		41	51	0	−
	B-cell enrichede		16	19	75	−
3	Total PBMCs	Seropositive	43	38	27	−
	T-cell enriched		47	43	12	−
	B-cell enriched		0	0	50	+
4	Total PBMCs	Seropositive	26	49	37	−
	T-cell enriched		19	59	12	−
	B-cell enriched		2.8	5.0	80	+
5	Total PBMCs	Seropositive	41	28	24	−
	T-cell enriched		47	35	15	−
	B-cell enriched		0	12	86	+

^aNegative selection by immunomagnetic bead depletion was performed according to the manufacturer’s instructions (Coulter-Immunotech). 

^bOne hundred thousand cells from each fraction were separated into five aliquots and stained individually with monoclonal antibodies labeled with FITC—OKT4 (CD4), B9.11 (CD8), and B-Ly-1 (CD20)—and the appropriate isotypic negative control. 

^cOne hundred thousand cells from each fraction were cocultured with primary rhesus fibroblasts for virus isolation in triplicate. A positive virus isolation was scored when two of three wells developed cytopathic effects characteristic of RRV (6, 13) after 10 days and confirmed by indirect immunofluorescence with serum samples obtained from monkeys experimentally inoculated with RRV strain 17577. 

^dThe T-cell-enriched population was generated by depleting PBMCs with B-Ly-1-conjugated magnetic beads (CD20⁺ cells; Coulter-Immunotech). 

^eThe B-cell-enriched population was generated by depleting PBMCs with the magnetic beads conjugated with the monoclonal antibodies FN18 (CD3⁺ cells; generously provided by M. Jonker) and B9.11 (CD8⁺ cells; Coulter-Immunotech).