Volume 10, Issue 5, Pages 503-513 (May 1999) Developmental Regulation of TCRδ Locus Accessibility and Expression by the TCRδ Enhancer  Robert J Monroe, Barry P Sleckman, Brianna C Monroe, Bernard Khor, Steven Claypool, Roger Ferrini, Laurie Davidson, Frederick W Alt  Immunity  Volume 10, Issue 5, Pages 503-513 (May 1999) DOI: 10.1016/S1074-7613(00)80050-3

Figure 1 Production of ES Cells and Mice with Targeted Deletions of Eδ (A) Schematic of the TCRα/δ locus (not to scale). BglII (“B”) and MspI (“M”) sites used for analysis of TCRδ rearrangements are shown. Also indicated with closed boxes are the Cα probe and probes 1, 3, 4, 6 (Cδ), and 8 (Livak et al. 1995). See text for other details. (B) The EδKO targeting construct is shown with schematics of a wild-type allele (“+”), an allele that has been targeted with the PGK-neor gene (“N”) or an allele with a single loxP site (“Δ”) replacing Eδ. loxP sites (shaded triangles); probes used for analysis of 5′ and 3′ homologous recombination (shaded boxes); and BamHI (“B”), NdeI (“N”), PstI (“P”), NheI (“Nh”), and ClaI (“C”) sites are indicated. (C) Southern blot with the 3′ probe (Cδ) on PstI-digested F1 ES cell DNA from wild-type cells (Eδ +/+) or cells heterozygous for Eδ replacement with the PGK-neor gene (Eδ +/N) or the loxP site (Eδ +/Δ). Targeting of the CBA versus C57BL6 alleles was distinguished by a PstI RFLP with the 3′ probe. (D) Southern blot carried out with the 3′ probe on PstI-digested tail DNA from Eδ +/+, Eδ +/Δ, and Eδ Δ/Δ littermates resulting from the breeding of Eδ +/Δ mice. Eδ +/Δ mice were generated by breeding germline Eδ +/N to EIIa-CreTG mice for the purpose of deleting the PGK-neor gene (data not shown). Immunity 1999 10, 503-513DOI: (10.1016/S1074-7613(00)80050-3)

Figure 2 Eδ Δ/Δ Mice Have Normal αβ T Cells but Reduced γδ T Cell Numbers (A) Thymocytes and splenocytes from Eδ +/+ and Eδ Δ/Δ littermates were stained with PE-conjugated anti-CD4 and FITC-conjugated anti-CD8 and analyzed by flow cytometry. Quadrant analysis of CD4−CD8−, CD4+CD8+, CD4+CD8−, and CD4−CD8+ populations (thymus) and of CD4+CD8− and CD4−CD8+ populations (spleen) is shown with the total number of thymocytes and splenocytes. A minimum of six 4- to 6-week-old Eδ +/+ and Eδ Δ/Δ mice were analyzed; data shown are from a representative experiment. (B) αβ T cells were purified from the spleens of Eδ +/+, Eδ +/Δ, and Eδ +/N mice. DNA isolated from these cells was digested with Eco0109I and analyzed by Southern blotting with probe 8 (see Figure 1A). All Vα to Jα rearrangements will delete the region 5′ of the Jα genes, which hybridizes to probe 8. The germline bands for the targeted (“T,” CBA) and nontargeted (“NT,” C57BL6) alleles are indicated. Eco0109I-digested kidney DNA from CBA, C57BL6, and CBA/C57BL6 mice is also shown. Hybridization to a Cα probe is shown as a DNA loading control. (C) Thymocytes and splenocytes from Eδ +/+, Eδ +/Δ, and Eδ Δ/Δ littermates were stained with FITC-conjugated anti-Thy1.2 and PE-conjugated anti-TCRδ chain antibodies and analyzed by flow cytometry. FACS data from representative experiments are shown with the percentage of γδ T cells (boxed regions) in each sample. (D) Numbers of γδ T cells per thymus and spleen of several Eδ +/+ (thymus, n = 8; spleen, n = 19), Eδ +/Δ (thymus, n = 13; spleen, n = 23), and Eδ Δ/Δ (thymus, n = 13; spleen, n = 26) mice were determined. The average number of cells for each phenotype is shown and indicated by a bar. Immunity 1999 10, 503-513DOI: (10.1016/S1074-7613(00)80050-3)

Figure 3 Eδ Is Required in cis for Normal Dδ to Jδ Rearrangement DNA from γδ T cells isolated from wild-type F1 mice (Eδ +/+) or mice heterozygous for the deletion of Eδ (Eδ +/Δ) was digested with BglII (panel A) or MspI (panel B) and subjected to Southern blot analysis using probe 4. MspI sites (“M”), BglII sites (“B”), and probe 4 are indicated in Figure 1A. (A) The germline BglII fragment (nonpolymorphic) is indicated (“GL”) in the kidney lane (C57BL6/CBA DNA). Rearrangements of upstream Vδ or Dδ1 gene segments to Dδ2 or Jδ1 result in loss of the germline and generation of novel BglII probe 4–hybridizing fragments. Residual germline hybridization in the Eδ +/+ lane (also in Figure 4B) is due to the presence of non-T cells (∼10%) in the Eδ +/+ γδ T cell preparation. A major probe 4–hybridizing band present in the Eδ +/Δ lane but not the Eδ +/+ lane is indicated with an arrow (“*”) and is the predicted size (5.5 kb) for a Dδ1-Dδ2 rearrangement. Hybridization to a RAG-2 probe is shown as a DNA loading control. (B) Probe 4 shows a MspI polymorphism between the C57BL6 (2.6 kb) and CBA alleles (2.3 kb). In Eδ +/Δ mice, the C57BL6 allele carries the nontargeted, wild-type δ enhancer (“NT”) while the CBA allele carries the targeted, loxP-replaced δ enhancer (“T”). The germline MspI fragments of both alleles are lost upon rearrangement of upstream Dδ1 or Dδ2 gene segments to the Jδ1 gene segment and replaced with novel probe 4–hybridizing fragments. Hybridization to a RAG-2 probe is shown as a DNA loading control and was used to normalize probe 4 hybridization to the CBA germline fragment (“T,” 2.3 kb). In brief, the ratio of probe 4 signal to RAG-2 signal (probe 4T:RAG-2) in each lane was calculated by densitometry. As the control sample does not undergo TCRδ rearrangement, the probe 4T:RAG-2 ratio for the kidney DNA lane was converted to a value of 1.0 and the ratios for the Eδ +/+ and Eδ +/Δ γδ T cell DNA lanes were adjusted by the same factor. Adjusted probe 4T:RAG-2 ratios are shown and represent the fraction of CBA alleles in each DNA sample which retain the germline Dδ2-Jδ1 configuration. Immunity 1999 10, 503-513DOI: (10.1016/S1074-7613(00)80050-3)

Figure 4 Production of ES Cells with Targeted Deletions of Eδ and Eα (A) Schematic of two alleles of the TCRα/δ locus, one with intact enhancers (B6) and one with targeted deletions of Eδ and Eα (CBA). LoxP sites (closed triangles), the Eα probe (closed box), and the StuI sites used for Southern blot analysis are indicated. A StuI RFLP allows B6 (3.2 kb) and CBA (2.3 kb) alleles to be distinguished. (B) Southern blot carried out with the 5′ Eα probe on HindIII-digested DNA from Eδ +/Δ ES cells targeted with the EαKO construct (Sleckman et al. 1997). Targeted replacement of Eα with the PGK-neor gene (Eα +/N) and a loxP site (Eα +/Δ) is shown next to an Eα +/+ control. (C) Southern blot carried out on StuI-digested DNA from C57BL6 kidney (B6), wild-type F1 ES cells (B6/CBA), and F1 ES cells heterozygous for the replacement of Eδ and Eα with loxP sites (EδΔEαΔ) with the Eα probe. The EδΔEαΔ lane shows a loss of Eα-hybridizing sequences on the CBA allele, shown previously to carry the EδΔ mutation (Figure 1B). Immunity 1999 10, 503-513DOI: (10.1016/S1074-7613(00)80050-3)

Figure 5 Eδ Is Required for Germline TCRδ Expression in DN Thymocytes but Not for TCRδ Expression in Mature γδ T Cells (A) Total cell RNA was harvested from thymocytes of Eδ Δ/Δ:RAG-2−/− and Eδ +/+:RAG-2−/− mice and analyzed by Northern blotting for TCRδ expression. The blot was probed with Cδ followed by Cγ1 and Cβ2 to normalize for DN thymocyte RNA. Values for the ratio of Cδ- to Cγ1-hybridizing RNA (Cδ:Cγ1) are shown as a measure of TCRδ expression. (B) Peripheral γδ T cells were purified from Eδ +/+, Eα Δ/Δ, and Eδ Δ/Δ mice. RNA from these cells (and from purified wild-type αβ T cells and RAG-2−/− spleen) was analyzed by Northern blotting with a Cδ probe to assess TCRδ expression. Subsequently, the blot was stripped and probed with a Cγ1 probe to normalize for γδ T cell RNA and GAPDH (“G”) to control for RNA loading. Values for the ratio of Cδ- to Cγ1-hybridizing RNA (Cδ:Cγ1) are shown as a measure of TCRδ expression. “ND,” not determined. Immunity 1999 10, 503-513DOI: (10.1016/S1074-7613(00)80050-3)