The TCR δ Repertoire in Normal Human Skin is Restricted and Distinct from the TCR δ Repertoire in the Peripheral Blood Wolfgang Holtmeier, Michael Pfänder,

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The TCR δ Repertoire in Normal Human Skin is Restricted and Distinct from the TCR δ Repertoire in the Peripheral Blood Wolfgang Holtmeier, Michael Pfänder, Andreas Hennemann, Wolfgang F. Caspary Journal of Investigative Dermatology Volume 116, Issue 2, Pages 275-280 (February 2001) DOI: 10.1046/j.1523-1747.2001.01250.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 CDR3 size spectratyping of TCR δ transcripts in normal human skin and PBMC. From five subjects (A-E) PCR-amplified TCRDV1 and TCRDV2 transcripts from two skin samples (I and II) as far as 2–10 cm apart and two PBMC samples (I and II) were run concurrently on the same gel. As shown, skin samples I and II analyzed for DV1 or DV2 yielded an almost identical, restricted band pattern that was distinct from that in the PBMC samples. Direct sequencing of dominant DV1 or DV2 bands of the same length in skin samples I and II, as indicated by the CDR3 length numbers to the left of the gels, revealed identical TCR δ transcripts. Numbers to the right of the gels indicate the CDR3 length of sequenced TCR δ transcripts from PBMC samples (see Figure 2). CDR3 lengths of translated TCR δ transcripts were calculated as described in Materials and Methods. Subjects A, B, and C had an excision of a nonmetastatic malignant melanoma and subjects D and E were operated for cosmetic reasons. Journal of Investigative Dermatology 2001 116, 275-280DOI: (10.1046/j.1523-1747.2001.01250.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 TCRDV1 and TCRDV2 junctional sequences from the skin and PBMC. Dominant bands from Figure 1 were excised, reamplified, and directly sequenced. The × in the fourth column indicate identical CDR3 rearrangements in the two skin samples and/or the two PBMC samples. For example, the sequence MP054 (first lane) from subject A with a CDR3 length of 14 was found in skin samples I and II. A total of 18 pairs with identical sequences in both skin samples could be identified. TCR δ rearrangements that were found in the skin and the peripheral blood are likely to be derived from circulating γ/δ T cells that were present in capillaries of the skin (e.g., sequence WE076, TCRDV1, subject B, CDR3 length 19). P nucleotides are underlined and germline sequences are indicated at the top in boldface type. Sequence data are available from EMBL/GenBank/DDBJ under the accession numbers AF312272 to AF7312282(DV1) and AF312283–AF312295(DV2). Journal of Investigative Dermatology 2001 116, 275-280DOI: (10.1046/j.1523-1747.2001.01250.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 CDR3 size spectratyping of TCR δ rearrangements amplified from genomic DNA. From two additional subjects (F and G) DNA was isolated from skin and PBMC and amplified with a TCRDV2 specific primer and a primer specific for the intron of TCRDJ1 or TCRDJ3. Similar to the amplification of cDNA (Figure 1), oligoclonal CDR3 profiles and identical nucleotide sequences (Figure 4) were obtained from the two skin samples, confirming that restricted CDR3 profiles are not secondary to highly activated γ/δ T cells producing high amounts of mRNA. Journal of Investigative Dermatology 2001 116, 275-280DOI: (10.1046/j.1523-1747.2001.01250.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 TCRDV2 junctional sequences derived from genomic DNA. Dominant bands from Figure 3 were excised, reamplified, and directly sequenced. Similar to TCR δ transcripts (Figure 2) identical TCR δ rearrangements could also be isolated from DNA preparations of the skin samples I and II. Sequence KS5291 (subject F, TCRDV2) with a CDR3 length 17 was out of frame as indicated by an asterisk. Sequence data are available from EMBL/GenBank/DDBJ under the accession numbers AF312296-AF312303. Journal of Investigative Dermatology 2001 116, 275-280DOI: (10.1046/j.1523-1747.2001.01250.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Amino acid sequences of cutaneous and peripheral TCR δ chains. (a) Translated TCR δ rearrangements from RNA (Figure 2) and (b) DNA preparations (Figure 4) are shown. Hydrophobic amino acids V, L, and I at codon 97 are highlighted in gray. Journal of Investigative Dermatology 2001 116, 275-280DOI: (10.1046/j.1523-1747.2001.01250.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions