The αβ T Cell Receptor Can Replace the γδ Receptor in the Development of γδ Lineage Cells  Ludovica Bruno, Hans Jörg Fehling, Harald von Boehmer  Immunity  Volume 5, Issue 4, Pages 343-352 (October 1996) DOI: 10.1016/S1074-7613(00)80260-5

Figure 1 Staining of Lymph Node Cells from Male H-2b (B6) αβ TCR Transgenic Mice Upper panel, surface Ig− (sIg−) cells are stained with T3.70 (specific for the transgenic αβ TCR) and CD8 antibodies. Gates show sorted populations (R2 for CD4−CD8−, T3.70+ and R3 for CD4−CD8low, T3.70+). Lower panels, reanalysis of sorted populations prior to fusion with the BW 100.129 cell line. Immunity 1996 5, 343-352DOI: (10.1016/S1074-7613(00)80260-5)

Figure 2 Expression of Endogenous and Transgenic TCRα Chain Message RNA from (A) CD4−CD8+, T3.70+ hybridomas from H-2b transgenic female mice, (B) CD4−CD8−, T3.70+ hybridomas, and (C) CD4−CD8low, T3.70+ hybridomas from H-2b transgenic male mice were separated on agarose–formaldehyde gels and transferred to nylon membranes. The filters were first hybridized to an oligonucleotide probe recognizing transcripts from endogenous TCRα chain genes (top panels), subsequently hybridized to a cDNA probe recognizing transcripts from the TCRα chain transgene (middle panels), and finally normalized with a β-actin probe (bottom panels). Numbers at the top of each section represent individual hybridomas. RNA from B6 and BALB/c lymph nodes (LN) served as positive and negative controls, respectively (the constant portion of the TCRα transgene was obtained from a BALB/c genomic library, and the transgene was introduced into B6 mice; the oligonucleotide probe used is complementary to an 18 bp insert present in the B6 constant region of the α locus, but absent in BALB/c strains of mice). BW was the thymoma used as fusion partner, in which part of the α locus is deleted; B6.2.16 was the T cell clone from which the transgenes were derived. The faint bands that are visible in some of the hybridomas after hybridization with the endogenous Cα-specific oligonucleotide (top panel in [C]) are smaller than those of functional TCRα message and have been previously described as sterile transcripts of the unrearranged Jα–Cα locus (Kronenberg et al. 1986). Immunity 1996 5, 343-352DOI: (10.1016/S1074-7613(00)80260-5)

Figure 2 Expression of Endogenous and Transgenic TCRα Chain Message RNA from (A) CD4−CD8+, T3.70+ hybridomas from H-2b transgenic female mice, (B) CD4−CD8−, T3.70+ hybridomas, and (C) CD4−CD8low, T3.70+ hybridomas from H-2b transgenic male mice were separated on agarose–formaldehyde gels and transferred to nylon membranes. The filters were first hybridized to an oligonucleotide probe recognizing transcripts from endogenous TCRα chain genes (top panels), subsequently hybridized to a cDNA probe recognizing transcripts from the TCRα chain transgene (middle panels), and finally normalized with a β-actin probe (bottom panels). Numbers at the top of each section represent individual hybridomas. RNA from B6 and BALB/c lymph nodes (LN) served as positive and negative controls, respectively (the constant portion of the TCRα transgene was obtained from a BALB/c genomic library, and the transgene was introduced into B6 mice; the oligonucleotide probe used is complementary to an 18 bp insert present in the B6 constant region of the α locus, but absent in BALB/c strains of mice). BW was the thymoma used as fusion partner, in which part of the α locus is deleted; B6.2.16 was the T cell clone from which the transgenes were derived. The faint bands that are visible in some of the hybridomas after hybridization with the endogenous Cα-specific oligonucleotide (top panel in [C]) are smaller than those of functional TCRα message and have been previously described as sterile transcripts of the unrearranged Jα–Cα locus (Kronenberg et al. 1986). Immunity 1996 5, 343-352DOI: (10.1016/S1074-7613(00)80260-5)

Figure 2 Expression of Endogenous and Transgenic TCRα Chain Message RNA from (A) CD4−CD8+, T3.70+ hybridomas from H-2b transgenic female mice, (B) CD4−CD8−, T3.70+ hybridomas, and (C) CD4−CD8low, T3.70+ hybridomas from H-2b transgenic male mice were separated on agarose–formaldehyde gels and transferred to nylon membranes. The filters were first hybridized to an oligonucleotide probe recognizing transcripts from endogenous TCRα chain genes (top panels), subsequently hybridized to a cDNA probe recognizing transcripts from the TCRα chain transgene (middle panels), and finally normalized with a β-actin probe (bottom panels). Numbers at the top of each section represent individual hybridomas. RNA from B6 and BALB/c lymph nodes (LN) served as positive and negative controls, respectively (the constant portion of the TCRα transgene was obtained from a BALB/c genomic library, and the transgene was introduced into B6 mice; the oligonucleotide probe used is complementary to an 18 bp insert present in the B6 constant region of the α locus, but absent in BALB/c strains of mice). BW was the thymoma used as fusion partner, in which part of the α locus is deleted; B6.2.16 was the T cell clone from which the transgenes were derived. The faint bands that are visible in some of the hybridomas after hybridization with the endogenous Cα-specific oligonucleotide (top panel in [C]) are smaller than those of functional TCRα message and have been previously described as sterile transcripts of the unrearranged Jα–Cα locus (Kronenberg et al. 1986). Immunity 1996 5, 343-352DOI: (10.1016/S1074-7613(00)80260-5)

Figure 3 Deletion of the δ Locus in Different Cell Subsets from Normal Mice Analysis of hybridomas derived from (left) CD4+CD8− and CD4−CD8+ (single positive), αβ+ (H57+) lymph node cells and (right) from CD4−CD8− (double negative), αβ+ thymocytes of (B6 × DBA/2) mice. Genomic DNA (10 μg) was obtained from individual hybridomas, digested with HindIII, and subsequently run on a 0.7% agarose gel. The top panels show Southern blots probed with a Cδ cDNA fragment. This probe is able to detect a polymorphism present in germline DNA of the two different strains, B6 and DBA/2 (see Figure 5 for details). The same Southern blots were subsequently hybridized with a TAP2-specific probe (bottom panels) to show DNA integrity. Numbers at the top of the blots represent individual hybridomas. The germline configuration of the Cδ locus in B6, DBA/2, and (B6 × DBA/2) mice is also shown. The DNA was extracted from a mixture of brain, kidney, and lung tissue. BW α−β− is the fusion partner. Immunity 1996 5, 343-352DOI: (10.1016/S1074-7613(00)80260-5)

Figure 4 Deletion of the δ Locus in Different Cell Subsets from TCRαβ Transgenic Mice Analysis of hybridomas derived from CD4−CD8+, T3.70+ lymph node cells of αβ TCR transgenic B6 female mice (left) or CD4−CD8−, T3.70+ and CD4−CD8low, T3.70+ lymph node cells from male αβ TCR transgenic mice (right). The Southern blots were performed as described in Figure 3 using the same Cδ probe. The Southern blot to the left was also probed with a TAP2 fragment. Numbers on top of the blots represent individual hybridomas. BW α−β− is the fusion partner. The germline configuration of the Cδ locus in (B6 × DBA/2)F1 and B6 mice is also shown. Immunity 1996 5, 343-352DOI: (10.1016/S1074-7613(00)80260-5)

Figure 5 Both Cδ Alleles Are Present in Hybridomas Derived from Lymph Node Cells of (B6 × DBA/2)F1 αβ Transgenic Male Mice (Left) Polymorphism of the Cδ locus in HindIII-digested genomic DNA from mouse strains B6 and DBA/2 and their F1 progeny (B6 × DBA/2). Genomic DNA was obtained from a mixture of brain, kidney, and lung tissue. The probe used was the same Cδ fragment as described in Figure 1. This probe detects bands of 0.6 and 2.0 kb in genomic DNA of B6 mice, bands of 0.6 and 1.7 kb in DNA from DBA/2 mice, and all three bands in (B6 × DBA/2)-derived DNA. The Cδ region is deleted in BW α−β−. (Right) Southern blot analysis of hybridomas derived from CD4−CD8−, T3.70+ (A–K) and CD4−CD8low, T3.70+ (2–32) lymph node cells of (B6 × DBA/2)F1 αβ TCR transgenic male mice. Top, Cδ probe; bottom, TAP2 probe. Immunity 1996 5, 343-352DOI: (10.1016/S1074-7613(00)80260-5)

Figure 6 Dδ–Jδ Rearrangements in Hybridomas Derived from CD4−CD8− and CD4−CD8low Lymph Node Cells of αβ TCR Transgenic Male Mice A Southern blot analysis of different hybridomas with two distinct probes specific for sequences within the Dδ–Jδ locus is shown. Hybridoma DNA was digested with PvuII (a) and PstI (b), respectively. The numbers at the top of the blots represent individual hybridomas. B6 germline and BW α−β− DNA is also shown. The schematic under the blots shows the location of the two probes in the Dδ–Jδ genomic region. Lanes 3–17, CD4−CD8− cells; lanes 1–13, CD4−CD8low cells. Immunity 1996 5, 343-352DOI: (10.1016/S1074-7613(00)80260-5)

Figure 7 TCRα and TCRγδ Surface Expression on Thymocytes and Lymph Node T Cells from TCRαβ Transgenic Mice Gates show sorted populations (TCRδ+ and TCRα+). Reanalysis of sorted populations by T3.70 and TCRδ antibodies as well as forward scatter (FSC) is shown for each organ on the right. Propidium iodide was used for gating on live cells only. All TCRδ+ cells were also TCRβ+ (data not shown). (A) shows pTα+/− female (left) and male (right) mice; (B) shows pTα−/− female and male mice. The following absolute cell numbers were obtained from the different TCRαβ transgenic mice: female pTα−/−, 30 × 106 thymocytes and 12.7 × 106 lymph node cells; female pTα+/−, 72 × 106 thymocytes and 14.1 × 106 lymph node cells; male pTα−/−, 3.2 × 106 thymocytes and 4.2 × 106 lymph node cells; male pTα+/−, 2.7 × 106 thymocytes and 8 × 106 lymph node cells. Immunity 1996 5, 343-352DOI: (10.1016/S1074-7613(00)80260-5)

Figure 7 TCRα and TCRγδ Surface Expression on Thymocytes and Lymph Node T Cells from TCRαβ Transgenic Mice Gates show sorted populations (TCRδ+ and TCRα+). Reanalysis of sorted populations by T3.70 and TCRδ antibodies as well as forward scatter (FSC) is shown for each organ on the right. Propidium iodide was used for gating on live cells only. All TCRδ+ cells were also TCRβ+ (data not shown). (A) shows pTα+/− female (left) and male (right) mice; (B) shows pTα−/− female and male mice. The following absolute cell numbers were obtained from the different TCRαβ transgenic mice: female pTα−/−, 30 × 106 thymocytes and 12.7 × 106 lymph node cells; female pTα+/−, 72 × 106 thymocytes and 14.1 × 106 lymph node cells; male pTα−/−, 3.2 × 106 thymocytes and 4.2 × 106 lymph node cells; male pTα+/−, 2.7 × 106 thymocytes and 8 × 106 lymph node cells. Immunity 1996 5, 343-352DOI: (10.1016/S1074-7613(00)80260-5)

Figure 8 TCRγδ Expression on Thymocytes from Male pTα−/−, TCRαβ Transgenic Mice Staining of total thymocytes of a male TCRαβ transgenic, pTα−/− mouse with anti-TCRδ (GL3) versus anti-Vγ2 (UC3-10A6), the predominantly expressed Vγ element in the adult thymus. Immunity 1996 5, 343-352DOI: (10.1016/S1074-7613(00)80260-5)

Figure 9 Surface Phenotype of Thymocytes from Male pTα−/−, TCRαβ Transgenic Mice Cells were stained by four colors with the following antibody combinations: (left) CD4, CD8, TCRα (T3.70), and TCRδ antibodies; (right, top and middle) CD4 plus CD8, TCRα, CD25, and CD44 antibodies; (right, bottom) CD4 plus CD8, TCRδ, CD25, and CD44 antibodies. The cells were gated according to the marker indicated at the top of the quadrants; double stainings with the antibodies indicated on the X and Y axes are shown. Immunity 1996 5, 343-352DOI: (10.1016/S1074-7613(00)80260-5)