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Blood, Vol. 111, Issue 12, 5734-5744, June 15, 2008
Keratinocyte growth factor and androgen blockade work in concert to protect against conditioning regimen-induced thymic epithelial damage and enhance T-cell reconstitution after murine bone marrow transplantation
Blood Kelly et al.
111: 5734
Supplemental materials for: Kelly et al
Files in this Data Supplement:
- Figure S1. Additive benefit of KGF pretreatment and androgen blockade on restoring thymopoiesis is maintained through day 56 post-BMT (JPG, 73.9 KB)
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Lethally irradiated B6 recipients of allogeneic (Balb/c) bone marrow were left untreated (BMT Control) or pretreated with KGF, Lupron or KGF+Lupron and analyzed for thymocyte cellularity at day 56 post-BMT alongside age/sex-matched, unmanipulated B6 controls (nonBMT Control). Data shown are mean absolute numbers ± SEM of (A) total thymocytes and of the thymocyte subsets: (B) CD8−CD4−double-negative, (C) CD8+CD4+ double-positive, (D) CD4+CD8−single-positive, and (E) CD4−CD8+ single-positive. The data are representative of 4 independent experiments with 4–5 mice per group; *, p<0.05 compared with untreated BMT recipients; #, p<0.05 compared with KGF-treated BMT recipients.
- Figure S2. Pretreatment with KGF and androgen blockade restore thymic architectural organization by day 26 post-BMT (JPG, 134 KB)
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Lethally irradiated B6 recipients of allogeneic (Balb/c) bone marrow were left untreated (BMT Control) or pretreated with KGF, Lupron or KGF+Lupron. (A–B) Immunofluorescence staining of thymic sections for the cytokeratins, K18 (red) and K5 (blue) identified mature cortical TEC (K18+K5−) within the cortical region of the thymus (“C”) and mature medullary TEC (K18−K5+) located within the medullary region of the thymus (“M”). These methods were used to assess maintenance of thymic architectural organization into distinct cortical and medullary regions in BMT recipients at (A) day 28 and (B) day 56 post-BMT. (i, iv) Thymic sections from an unmanipulated, age/sex-matched B6 control (nonBMT Control) illustrates a clear distinction between cortex and medulla. (ii) BMT controls show a loss of this clear compartmentalization while (iii) KGF−, (iv) Lupron− and (v) KGF+Lupron-treated BMT recipients demonstrate thymic architectural organization similar to nonBMT controls. (B, vi-x) By day 56 post-BMT all groups exhibited similar architectural organization, i.e., corticomedullary distinction, compared with nonBMT controls. Data are representative of two experiments with 3 mice per group. Images were acquired on an Olympus FV500 confocal microscope using10×/0.40 objective lens with associated Olympus Software and processed with Adobe Photoshop.
- Figure S3. Combined pretreatment with KGF and androgen blockade significantly restore numbers of total and donor-derived, naïve CD4+ and CD8+ T cells in lymph node and spleen through day 60 post-BMT (JPG, 120 KB)
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Lethally irradiated B6 recipients of allogeneic (Balb/c) bone marrow were left untreated (BMT Control) or pretreated with KGF, Lupron or KGF+Lupron and analyzed for the presence of T cells in the lymph nodes and spleen at day 60 post-BMT alongside unmanipulated age/sex-matched B6 controls (nonBMT Control). Mean absolute numbers ± SEM of (A,E) total CD4+ T cells, (B,F) naïve (CD45RBhighCD44low) CD4+ T cells, (C,G) total CD8+ T cells, and (D,H) naïve (CD62LhighCD44low) CD8+ T cells in the lymph nodes and spleen are shown. Lymph node data is represented here by pooled cells from 2 inguinal, 2 axillary and mesenteric lymph nodes. Data are representative of 3 experiments, each with 4 mice per group; *, p<0.05 compared with BMT controls; #, p<0.05 compared with KGF-treated BMT recipients.
- Figure S4. KGF treatment and androgen blockade prior to BMT does not affect diversity of TCR Vβ expression in thymus-derived T cells post-BMT (JPG, 73.3 KB)
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Lethally irradiated C57BL/6 Ly5.2+ recipients of congenic C57BL/6 Ly5.1+ bone marrow were left untreated (BMT Control) or pretreated with KGF, Lupron or KGF+Lupron. Blood samples taken from BMT recipients and age/sex-matched, unmanipulated male C57BL/6 controls (nonBMT Control) were stained with monoclonal antibodies against Ly5.1 (donor), CD3 and TCR V at day 60 post-BMT. The percentage of V-positive cells among donor-derived CD3+ lymphocytes is shown. TCR V17ais an indicator of intact negative selection as this TCR is generally deleted in an intact thymus of C57BL/6 mice and is therefore detected at very low frequency in the nonBMT controls. Data shown are mean ± SEM from one experiment of 5 mice per group.
- Figure S5. Combined pretreatment with KGF and androgen blockade prior to BMT significantly enhances immune-mediated clearance of Listeria monocytogenes after allogeneic BMT (JPG, 58.3 KB)
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Lethally irradiated (Balb/c × B6)F1 recipients of allogeneic (Balb/c) bone marrow were left untreated (BMT Control) or pretreated with KGF, Lupron or KGF+Lupron and immunized at day 42 post-BMT alongside unmanipulated age/sex-matched B6 controls (nonBMT Control) with 105 CFU of L. monocytogenes strain 2C. After 28 days, mice were rechallenged with 2 × 106 CFU and livers were collected 4 days after secondary infection. Bacterial CFU were determined by plating of serial dilutions of organ homogenates onto BHI agar. Baseline for the y-axis was set to indicate the limit of detection (L.O.D., approximately 100 organisms) for this assay. Data are representative of two independent experiments, each with 4–5 mice per group and time point; *, p<0.05 compared with BMT controls.
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