CXCR4 regulates β-selection in vivo. (a) CXCR4 surface expression (blue) on DN thymocyte subsets. Grey histograms show staining with isotype-matched control antibody (n>5). (b) Quantitative PCR analysis of Cxcr4 and Cxcr7 expression in DN thymocyte subsets from C57BL/6 mice. Transcript expression was normalized to Gapdh within each subset (n=2). (c) C57BL/6 mice were injected intraperitoneally with saline or the CXCR4 antagonist AMD3100 (every 48 hours for a period of 7 days. A representative flow cytometric profile of DN subsets from one of five mice is shown with the average thymic cellularity indicated above each dot plot (n=2). (d) Thymi from Lck-Cre⁺ Cxcr4^+/+ and Lck-Cre/Cxcr4^fl/fl mice were isolated. Total cellularity (above dot plots) and surface marker expression were determined. Lower dot plots are gated on DN thymocytes. n>16 for each genotype. (e) Absolute numbers of thymic subsets in Lck-Cre⁺ Cxcr4^+/+ and Lck-Cre⁺ Cxcr4^fl/fl mice. Data show the mean ± s.d. of at least ten littermates for each genotype (4-6 weeks old). Littermates were obtained from 3 or more different breeding cages.

CXCR4 provides differentiation and survival signals during T cell development. (a) Purified DN3e thymocytes from the Lck-Cre⁺ Cxcr4^fl/fl and Lck-Cre⁺ Cxcr4^+/+ mice (small CD4⁻CD8⁻CD3⁻CD25⁺ thymocytes that were electronically sorted based on their size, after gating out cells expressing any of a panel of hematopoietic lineage markers) were seeded on OP9-DL1 stromal cells. The differentiation to later stages of DN and DP development was determined on days 2, 4 and 8 post-initiation of co-culture. The bottom panels are gated on total thymocytes whereas all other panels are gated on DN cells (n=3). (b) The absolute numbers of total DN3 thymocytes (left panel) or total DN and DN4 (right panel) in the cultures described in a were determined by flow cytometry. Each co-culture was done in triplicates and s.e.m. reflects variation. (c) Sca1⁺ c-kit⁺ bone marrow progenitors from indicated mice were electronically sorted based on a panel of hematopoietic lineage markers. Purified hematopoietic progenitors were then seeded on OP9-DL1 stromal cells. Differentiation to later stages of development was determined by flow cytometry on days 14, 16 and 22 post-initiation of co-culture (n=2). (d) Absolute numbers of cells present in cultures described in c. Each co-culture was done in triplicates and s.e.m. reflects variation. (e) c-kit⁺ immature thymocytes (DN1) from indicated mice were sorted and seeded on OP9-DL1 cultures and analyzed by flow cytometry on day 3 and 6. Top plots are gated on DN cells and bottom plots are gated on total thymocytes (n=2). (f) Absolute numbers of cells present in cultures described in e. Each co-culture was done in triplicates and s.e.m. reflects variation. (g) Purified DN3e cells were seeded on OP9-DL1 cultures in the presence or absence of the CXCR4 antagonist AMD3100, and absolute cell numbers were measured at the indicated time points. Each co-culture was done in triplicates and s.e.m. reflects variation. (n>3).

(a) Thymi from Lck-Cre⁺ Cxcr4^+/+ and Lck-Cre⁺ Cxcr4^fl/fl mice expressing the DO11.1 TCR transgene were isolated. Absolute thymocyte numbers were quantified and surface marker expression was assessed by flow cytometry. Top dot plots were gated on total thymocytes and bottom dot plots on DN cells (n=3). (b) Absolute numbers of DN3 and DN4 thymocytes in mice analyzed in a were calculated by multiplying total thymocyte numbers by the percent distribution of different thymic subsets. Data were plotted as the mean ± s.d. of 4-6 week old littermates for each genotype, obtained from three or more different breeding cages. (c) DO11.10 TCR surface expression on the indicated thymocyte subsets was determined using the clonotypic KJ126 antibody. (d) Lck-Cre⁺ Cxcr4^+/+ and Lck-Cre⁺ Cxcr4^fl/fl mice were intraperitoneally injected with saline or anti-CD3. Forty-eight hours later, thymi were collected, single cell suspensions were counted and stained for the indicated differentiation markers. Dot plots are gated on DN thymocytes (n=2). (e) To assess cell proliferation, DN thymocytes from Lck-Cre⁺ Cxcr4^+/+ and Lck-Cre⁺ Cxcr4^fl/fl mice were labeled with CFSE and cultured with OP9-DL1 stromal cells. CFSE dilution was measured at the indicated time points by flow cytometry. (n=2).

ShcA is an essential player downstream of CXCR4 signaling for migration and localization of DN thymocytes. (a) SCIET27 and SCB29 cells were stimulated for the indicated times with SDF-1α, or with anti-CD3 (positive control). Tyrosine phosphorylation of SchA was assessed by immunoblotting. (n=3). (b) Thymocytes from Shc1^fl/fl and Lck-Cre+Shc1^fl/fl mice (four mice for each group) were stimulated or not with SDF-1α, and chemotaxis through laminin or BSA-coated cells was measured. Graphs show the percentage of migrated DN3 cells compared to input. (n=4). (c) Thymocytes from indicated mice were stimulated with SDF-1α, and F-actin polymerization was measured at the indicated time points. (n=2). (d) Purified indo-1 labeled DN thymocytes were stimulated with SDF1-α and calcium flux was measured using a fluorimeter. Where indicated, thymocytes were pre-incubated with EGTA. (n=2). (e) CXCR4 expression on the surface of thymocyte subsets was measured by flow cytometry. Light gray histogram indicates isotype control antibody staining. DP thymocytes from the Lck-Cre⁺ Shc1^fl/fl mice served as a control since these represent ‘normal’ thymocytes that escaped Cre-mediated deletion43. (n>3). (f) Thymic cryosections from Shc1^fl/fl and Lck-Cre⁺ Shc1^fl/fl littermates were stained for CD25 (red) and SDF-1α (green). Original magnification ×10. (n=6). (g) Average CD25 pixel intensities derived from scanning serial sections shown in f (three mice per each group).

CXCR4 deletion affects localization and survival of early thymic progenitors in vivo. (a) Thymic cryosections from Lck-Cre⁺ Cxcr4^+/+ and Lck-Cre⁺ Cxcr4^fl/flmice (4-6 weeks old) were stained with antibodies to CD25 (red) and SDF-1α (green). (n=6). (b) The average CD25 pixel intensities within the subcapsular zone (SCZ), cortex and cortico-medullary junction (CMJ) were quantified by scanning multiple serial sections from Lck-Cre⁺ Cxcr4^+/+ and Lck-Cre⁺ Cxcr4^fl/flmice (three mice per group) (n=2). (c) Thymic sections from Lck-Cre⁺ Cxcr4^+/+ and Lck-Cre⁺ Cxcr4^fll/fl mice were stained for fragmented nuclei from apoptotic thymocytes (brown, apostain) and counterstained with hematoxylin (blue background tissue staining). Images show apoptotic nuclei from the SCZ (top and middle panels) and the medulla (bottom panels). n=2. Top and bottom panels, original magnification ×20. Middle panel shows boxed region in top panels, original magnification ×40. (n=2). (d) Apoptotic nuclei in the cortex and at the SCZ were counted from multiple sections and thymi from at least two independent staining out of the same tissues.. Data were plotted as the mean mean ± s.d.

CXCR4 is required for pre-TCR dependent survival signals. (a) Cell death was measured as percentages of DN3 thymocytes from the Lck-Cre⁺ Cxcr4^+/+ and Lck-Cre⁺ Cxcr4^fl/fl that become annexin V-positive in OP9-DL1 co-cultures on the indicated days (n=2). (b) Representative histogram showing profile of annexin V staining of cells described in a on day 2 and day 6 (n=2). (c) Bcl2a1 mRNA expression in DN3e, DN3L and DN4 thymocytes sorted from Lck-Cre⁺ Cxcr4^+/+ and Lck-Cre⁺ Cxcr4^fl/fl mice was measured by quantitative PCR. Expression was normalized to Gapdh, and the DN3e population from Lck-Cre⁺ Cxcr4^+/+ mice was set as the arbitrary unit of 1 to allow relative quantification (n>3). (d) Bcl2a1 mRNA expression was tested by quantitative PCR in purified DN cells from PBS or anti-CD3 treated Lck-Cre⁺ Cxcr4^+/+ and Lck-Cre⁺ Cxcr4^fl/fl mice. The saline-injected population from Lck-Cre⁺ Cxcr4^+/+ mice was set as the arbitrary unit of 1 to allow relative quantification (n=2).

The pre-TCR regulates CXCR4 signaling and physically associates with CXCR4. (a) Purified DN thymocytes from C57BL/6, Rag2^−/− and Rag2^−/− TCRβ⁺ mice were tested for chemotaxis to 10nM SDF-1α or bovine serum albumin (BSA) (n=5). (b) Migration of pre-T cell lines SCIET27 (pre-TCR-deficient) and SCB29 (pre-TCR-expressing) to 20nM SDF-1α or BSA (n=5). Data in a and b are presented as the percentage of input cells that migrate to SDF-1α. (c) CXCR4 surface expression on thymocytes from Rag2^−/− and Rag2^−/−TCRβ⁺ mice and SCIET27 and SCB29 cells was measured by flow cytometry. Light grey histograms indicate isotype control antibody (n=3). (d) SCIET27 and SCB29 cells were stimulated for the indicated time periods with SDF-1α. F-actin polymerization was analyzed by flow cytometry using Alexa-647-labeled phalloidin. Data show MFI of phalloidin staining. n=2. (e) SCB29 cells were stimulated for the indicated time periods with SDF-1α, and staining for CXCR4 (green) and pre-TCR (red) was analyzed by confocal microscopy. The images shown are representative of multiple cells on the same slide with similar phenotype (n=2). White arrowheads denote close interaction between pre-TCR and CXCR4. (f) SCIET27 and SCB29 cells were stimulated for the indicated times with SDF-1α and Erk phosphorylation was measured by immunoblotting. Red numbers indicate densitometry analysis of Erk phosphorylation normalized to total Erk expression. (n=5). (g) DN3 thymocytes from C57BL/6 mice were treated with a membrane-permeable Erk inhibitory peptide (iErk), the CXCR4 antagonist AMD3100 or the calcium chelators EGTA and BAPTA. Migration to SDF-1α was then assessed using Transwells. Bar graphs show percentage of migrated DN3 cells as a fraction of total cell input, ± s.d. (n=2). (h) DN3 thymocytes pre-treated with iErk or a control Flag peptide or vehicle alone, and were stimulated with SDF-1α. F-actin polymerization was assessed by flow cytometry. (n=2). (i) SCIET27 and SCB29 cell lines were stimulated with SDF-1α and calcium flux was measured by spectrofluorimetry (n=2). (j) SCB29 cells were left untreated or were treated with the calcium chelators EGTA or BAPTA. F-actin polymerization after SDF-1α stimulation was assessed by flow cytometry. (n>2).