1. T-Cell Receptor Vα Usage by Effector CD4+Vβ11+ T Cells Mediating Graft-versus-Host Disease Directed to Minor Histocompatibility Antigens 
Christine G. DiRienzo, George F. Murphy, Thea M. Friedman, Robert Korngold 
Biology of Blood and Marrow Transplantation 
Volume 13, Issue 3, Pages (March 2007)
DOI: /j.bbmt
Copyright © 2007 American Society for Blood and Marrow Transplantation Terms and Conditions

2. Figure 1
Evolution of B6→BALB.B GVHD in lingual epithelium, days 10 and 15. (A) On day 10, hematoxylin and eosin histology reveals lymphocytes within the superficial submucosa and epithelium. The small, somewhat hyperchromatic lymphocyte nuclei can be easily differentiated from the larger, more open nuclei of the squamous mucosal or columnar epithelial target cells they are infiltrating. (B) The lymphocytes increase in number by day 15, when they become more concentrated at the rete-like ridges and associated with target cell apoptosis (arrow; inset is B6→B6 d15 control). (C, D) Immunohistochemistry demonstrates that > 90% of these lymphocytes express Vβ11. Note the tendency of these cells to associate with target cells in the basal layer by day 15 [arrow in (D); the inset represents negative staining for Vβ5 in adjacent section]. (E, F) The B6→B6 control tongue failed to show Vβ11 infiltration at day 10 (E) and day 15 (F).
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2007 American Society for Blood and Marrow Transplantation Terms and Conditions

3. Figure 2
Evolution of B6→BALB.B GVHD in intestinal epithelium, days 10 and 15. Pathological changes are not apparent at day 10 (A) but are noted by day 15 (B), consisting of lymphoid infiltration and apoptosis of crypt epithelium (arrows). The inset in (B) depicts day 15 B6→B6 control. (C) Rare Vβ11+ cells are present at day 10 (arrow), whereas numerous positive cells are present at day 15 (D). In (D), the inset is staining for Vβ5 in an adjacent section. The B6→B6 control gut fails to show significant infiltration by Vβ11-positive cells at either day 10 (E) or day 15 (F).
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2007 American Society for Blood and Marrow Transplantation Terms and Conditions

4. Figure 3
Isolation of responding B6 CD4+Vβ11+ T cells. B6 mice were serially immunized with BALB.B splenocytes (2 × 107, IP) on days 0, 17, and 42. On day 63, spleen and LN CD4+ T cells from these mice were labeled with CFSE and cultured for 3 days with either L-mimosin to distinguish nondividing cells (A) or irradiated BALB.B splenocytes that were depleted of all T cells (B). Histograms, gated on CFSE+CD4+Vβ11+ T cells, indicated that most of the allostimulated cells underwent division, marked by decreased CFSE intensity.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2007 American Society for Blood and Marrow Transplantation Terms and Conditions

5. Figure 4
Alignment of TRAV sequences isolated from CFSE-labeled B6 CD4+Vβ11+ T cells. After RNA extraction from CFSElow-negative CD4+Vβ11+ T cells, cDNA was enriched for TCRα chain transcripts using a NotI-Cα3 primer and subsequently amplified using NPA-PCR. Products were visualized on an agarose gel, gel-purified, then cloned into a pGEM T-Easy vector. DNA sequences isolated from bacteria colonies and sequences were aligned for similarity using the CLUSTALW program. All sequences were compared to those found in the IMGT database using the IMGT/V-QUEST program. (A) Sequence alignment of all sequences, in frame and out of frame. All sequences were compared against CD-D16, which was used as a reference sequence for comparison with the other sequences for similarity or differences. Dashes represent nucleotide identity, and dots represent nucleotide gaps. Nucleotides that differ from the reference sequence have the letters written in the areas that differ. CD-D16, CD-D14, CD-D2, CD-D6, and CD-D10 contain the sequences for TRAV13-TRAJ16. CD-D12, CD-D37 CD-D1, CD-D3, and CD-D4 contain the sequences with TRAJ17. CD-D9 contains an out-of-frame sequence for TRAV13–TRAJ43. CD-D13 is an incomplete sequence and contains only TRAJ and TRAC regions. (B) TRAV13–TRAJ16. Note that the amino acids are included above the nucleotide sequences in the alignment. TRAV, N (N-region additions), TRAJ, and TRAC regions are indicated above nucleotides.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2007 American Society for Blood and Marrow Transplantation Terms and Conditions

6. Figure 5
LMD targeting of B6 Vβ11+ T cells in BALB.B organs. B6 CD4+ T cells (2 × 107) and ATBM (2 × 106) were transferred into lethally irradiated (10 Gy) BALB.B or control B6 recipients. Immunohistochemistry of uncoverslipped tissue sections stained with Vβ11 antibody and counterstained with hematoxylin. (A, B) LMD-targeted areas of lingual epithelium RLP before (A) and after (B) LMD. (C, D) Targeted areas of small intestines before (C) and after (D) LMD. Control mice were lethally irradiated (10 Gy) and injected with 2 × 106 B6 ATBM. (E, F) RLP of control mice before (E) and after (F) LMD. (G, H) Targeted intestinal areas of control mice before (G) and after (H) LMD. Note the areas that have been extracted by LMD. The pictures are from 1 mouse and are representative of 2 separate experiments, each with n = 3.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2007 American Society for Blood and Marrow Transplantation Terms and Conditions

7. Figure 6
PCR amplification of TRAV isolated by LMD. Amplified products were visualized on 3% agarose gels. CD4 (285bp) and TRAV13–TRAJ16 (69 bp)-specific primers were used to amplify DNA from LMD-extracted lingual (A and C) and intestinal epithelium (B and D) 15 days after transplantation of either 2 × 107 B6 CD4+ T cells and 2 × 106 ATBM into BALB.B recipients (10 Gy) (A and B) or 2 × 106 B6 ATBM into B6 recipients (10 Gy) (C and D). For each gel, lane 1 is a 100-bp ladder, and lanes 2-4 are each amplified products from a single mouse and are representative of 2 separate experiments (n = 6).
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2007 American Society for Blood and Marrow Transplantation Terms and Conditions