Volume 4, Issue 1, Pages 47-55 (January 1996) Single-Cell PCR Analysis of TCR Repertoires Selected by Antigen In Vivo: A High Magnitude CD8 Response Is Comprised of Very Few Clones  Janet L. Maryanski, C.Victor Jongeneel, Philipp Bucher, Jean-Laurent Casanova, Paul R. Walker  Immunity  Volume 4, Issue 1, Pages 47-55 (January 1996) DOI: 10.1016/S1074-7613(00)80297-6

Figure 1 Flow Cytometric Sorting of Single Cells The staining patterns and sorting schemes are shown for PBL from a representative immune mouse (A) and for PBL from nonimmunized control mice (B). The cells were stained with MAbs specific for Vβ10, CD62L, and CD8. The subpopulations gated as shown were sorted as single cells into PCR tubes. Immune PBL were sorted as Vβ10+CD62L−CD8+ cells. Normal PBL were first sorted as a pool of CD8+ cells, and these were then sorted as single Vβ10+CD8+ cells. Immunity 1996 4, 47-55DOI: (10.1016/S1074-7613(00)80297-6)

Figure 2 Vβ10–Jβ1.2 TCRs Expressed by Single Vβ10+CD62L− CD8 T Cells Sorted from Individual CW3-Immune Mice DBA/2 mice (5) were injected twice with P815-CW3 transfectant cells over a 2 month interval. The mice were bled 15 days after the first injection and 12 days after the second injection, corresponding to primary and secondary responses, respectively. PBL were stained, and single Vβ10+CD62L− CD8 T cells were sorted into individual PCR tubes for amplification with Vβ10 and Jβ1.2 primers, as described in Experimental Procedures. The sequences shown represent the deduced amino acid sequences of the CDR3 regions beginning with residue 95 (Chothia et al. 1988) of amplified PCR products that were rearranged to the Jβ1.2 gene segment. The nucleotide sequences (NS) are shown in Figure 7. For each mouse, identical amino acid sequences found in both the primary and secondary responses and encoded by the same NS are shown on the same line. Both the number of cells found to express an identical NS for a given animal within the same response (primary or secondary), and its calculated percent of the Jβ1.2 sequences amplified are shown to the right of the relevant CDR3 sequences. For each set of data, the proportion amplified is shown both as a fraction indicating the number of Jβ1.2 PCR products obtained out of the total number of cells analyzed, and as a calculated percentage. Immunity 1996 4, 47-55DOI: (10.1016/S1074-7613(00)80297-6)

Figure 3 CDR3 Sequences of Vβ10–Jβ1.2 TCRs Expressed by CW3-Specific CTL Clones The β chain CDR3 regions of previously characterized (Casanova et al. 1992; Casanova et al. 1993) and additional (PED 3, PED 11) CW3-specific CTL clones that express Vβ10–Jβ1.2 TCRs is shown. The clones are considered independent either because they originate from different mice, or if from the same pool of mice they express distinct NS (Figure 7). Immunity 1996 4, 47-55DOI: (10.1016/S1074-7613(00)80297-6)

Figure 7 Distinct Vβ10–Jβ1.2 Nucleotide Sequences of TCRs Expressed by CW3-Specific CTL Clones and Single Vβ10+CD62L− CD8 T Cells Sorted from CW3-Immune Mice The nucleotide sequences correspond to the V–D–J region of Vβ10–Jβ1.2 TCRs amplified from single CW3-immune cells (Figure 2), or expressed by previously described and additional CW3-specific CTL clones (Casanova et al. 1992; Casanova et al. 1993; Figure 3). For convenience, sequences encoding CDR3 lengths of 6 aa or 7 aa are shown in (A) and (B), respectively. NS refers to the nucleotide sequence code defined in Experimental Procedures. The consensus amino acid sequence beginning with residue 93 (according toChothia et al. 1988) is indicated at the top, and the deduced CDR3 region sequence (beginning with residue 95) for each NS is shown on the right side. The germline sequences for Vβ10 (Hirama et al. 1991) and Jβ1.2 (Gascoigne et al. 1984) are shown below each series. Immunity 1996 4, 47-55DOI: (10.1016/S1074-7613(00)80297-6)

Figure 4 Small Size of the Vβ10–Jβ1.2 TCR Repertoire Expanded in Individual CW3-Immune Mice The median estimate (X) and 95% confidence intervals of the size of the Vβ10-Jβ1.2 TCR repertoire is shown for the primary (1°) and secondary (2°) responses of individual mice (numbers 1–5), except for mouse 4, where only the primary response was analyzed. The calculations for hyperimmunized mouse 5 are also shown (4°). These were calculated as described in Experimental Procedures using the data shown in Figure 2. Immunity 1996 4, 47-55DOI: (10.1016/S1074-7613(00)80297-6)

Figure 5 Vβ10–Jβ1.1 TCRs Amplified from CW3-Immune Mice Sequences amplified during the same experiments presented in Figure 2, but found to express a Vβ10 TCR rearranged to the Jβ1.1 gene segment instead of Jβ1.2 are shown. Immunity 1996 4, 47-55DOI: (10.1016/S1074-7613(00)80297-6)

Figure 6 Vβ10 TCRs Amplified from Nonimmune DBA/2 Mice Single Vβ10+ CD8 T cells sorted from normal DBA/2 mice were amplified with the same primers used for the experiments in Figure 1 and Figure 4. The CDR3 sequences of the 13 PCR products amplified from 188 total cells are shown. The Jβ segments were assigned according to known genomic sequences (Kavaler et al. 1984; Siu et al. 1984). Immunity 1996 4, 47-55DOI: (10.1016/S1074-7613(00)80297-6)