. 2003 Mar 28;100(8):4730–4735. doi: 10.1073/pnas.2627966100

Table 1.

Sequence analysis of Ig gene rearrangements

Case	Cell type	Location	No. of EBV PCR-positive samples/samples analyzed^*	No. of V genes			% of cells (rearrangements^†) with mutated V genes^‡	% of cells (rearrangements^†) assigned to clones	Clones with mutated V genes/total no. of clones	Intraclonal V gene diversity
Case	Cell type	Location	No. of EBV PCR-positive samples/samples analyzed^*	V_H	V_κ	V_λ	% of cells (rearrangements^†) with mutated V genes^‡	% of cells (rearrangements^†) assigned to clones	Clones with mutated V genes/total no. of clones	Intraclonal V gene diversity
1	EBER⁺	GC1	30 /47	16	1		100	94	1 /1
		IFR^§	47 /68	18			100	100	1 /1
		All	77 /115	34	1		100	97	1 /1^¶	No
2		GC2	11 /32	4	4	4	84	67	3 /3
		GC3	17 /28	7	11	5	100	73	6 /6
		IFR^§	114 /180	59	40	34	95	63	15 /15
		All	142 /240	70	55	43	95	64^∥	17 /17^¶	No
1	EBER^−†	GC1	0 /45	13	16	11	66	20	3 /3	Yes
2		GC2	0 /38	19	12	9	91	50^∥	6 /6	Yes
		GC3	3 /40	9	11	5	42	4	0 /1^**	?
		Mantle of GC2	0 /22	14	12	12	10	0	0 /0
		Mantle of GC3	0 /16	8	10	7	0	8	0 /1	No
Controls
1	B cells^‡‡		11 /23	7	6	7	100	46	1 /1	No
	T cells^‡‡		4 /36	4^††	1^††	1^††
	Buffer		0 /41	0	0	0
2	Buffer		0 /59	0	0	0

Samples of case 1 and case 2 were analyzed for the presence of a fragment of the EBNA1 and LF2 gene, respectively. For EBV⁺ cells the efficiencies of EBV-specific PCRs are expected to vary between 40% and 70% because of technical matters such as DNA instability and loss of cells during transfer into reaction tubes.

^†

Because groups of three to five EBER⁻ cells were isolated, numbers given for these samples refer to the number of rearrangements amplified.

^‡

Disregarding nonfunctional V_κ gene rearrangements, which are usually inactivated and hence exempted from somatic hypermutation.

^§

Including some EBER⁺ cells located at the border of GCs. To avoid wrong assignment of these cells to the GCs, they were counted as cells from the IFR.

^¶

Members of individual clones were located inside, as well as outside, GCs.

^‖

From four members of two clones, unique V gene rearrangements were amplified that are incompatible with the clonal rearrangements obtained from other cells of these clones, indicating a low level of cellular contamination. These samples were not taken into further consideration.

^**

For one clone consisting of a member isolated from GC2 and a member from GC3, only one member harbored somatic mutations in the clonal V gene rearrangement.

^‡‡

CD20⁺ B cells and CD3⁺ T cells were isolated from sections adjacent to sections from which EBER⁺ and EBER⁻ cells were micromanipulated.

^††

The four V_H and the two V_λ gene rearrangements originate from four T cell samples. Although this indicates a low level of cellular contamination in the analysis, the reliability of the results is demonstrated by (i) the higher frequency of PCR products from EBER⁺ cells (30–43%) compared with the T cell samples (11%, data not shown), (ii) the lack of PCR products from 100 buffer controls, (iii) the very low frequency of EBV PCR products (2%) obtained from EBER⁻ samples, and (iv) the fact that all V gene PCR-positive samples of EBER⁺ cells from case 1 were indeed positive in the EBV PCR.