1. Functional divergence of antigen-specific T-lymphocyte responses in syngeneic graft- versus-host disease 
Christopher J Thoburn, Yuji Miura, Emilie C Bright, Allan D Hess 
Biology of Blood and Marrow Transplantation 
Volume 10, Issue 9, Pages (September 2004)
DOI: /j.bbmt

2. Figure 1
CLIP autoreactive T-cell subsets. Spleen cells from animals with SGVHD were harvested during acute and chronic SGVHD and assessed for killer-cell activity by using the 3H-thymidine assay [16,22]. Before graded numbers of effector T lymphocytes were added, the target cells were loaded (displacing natively expressed CLIP) with the CLIP variants that, in addition to the MHC class II-binding domain, include either the N- or C-terminal flanking domains or neither domain. Untreated target cells express native CLIP that includes both terminal-flanking domains. Results are expressed as the percentage of specific killing ± SD. The N-terminal CLIP preference of cells isolated during acute SGVHD was significantly different (P < .01, analysis of variance) compared with the C-terminal restriction profile of the cells assessed during chronic SGVHD. The shaded area, however, represents the effector-target ratio that provides the greatest discrimination between N- and C-terminal flanking domain dependence.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )

3. Figure 2
Flow cytometric selection of CLIP antigen-specific T-cell subsets. Splenic T lymphocytes harvested on day 14 after cessation of cyclosporine treatment were subjected to 3-color immunofluorescence. The cells were stained with the biotinylated soluble MHC class II immunoglobulin construct loaded with the N- or C-terminal CLIP variants (counterstained with CyChrome/streptavidin) and with fluorescein isothiocyanate (FITC)-and phycoerythrin (PE)-conjugated antibodies to CD8 and CD25, respectively. The percentage of cells staining with the construct is shown in the histogram; the dot plot displays CD8 and CD25 expression of the cells reacting with the peptide-loaded construct.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )

4. Figure 3
Differential function and specificity of the CLIP-reactive T cells in acute and chronic SGVHD. Splenic lymphocytes isolated from animals with acute and chronic SGVHD (harvested on days 7 and 42, respectively, after cessation of cyclosporine treatment) were left unfractionated or were separated flow cytometrically on the basis of reactivity with the soluble MHC class II immunoglobulin construct loaded with the N- or C-terminal CLIP variants. Before assay, the cells were extensively washed in complete tissue culture medium. The lymphocyte subsets were assessed for their ability to kill target cells loaded with the CLIP variants (3H-thymidine-based killer cell assay) at a 15:1 effector-target cell ratio; the results are expressed as the percentage of specific killing ± SD. The cells were also assessed for cytokine mRNA levels by quantitative PCR normalized against mRNA transcript levels for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). BD indicates binding domain. ∗∗∗No mRNA transcripts were detected. The results of a representative experiment are presented.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )

5. Figure 4
Localized cytokine mRNA transcription in acute and chronic SGVHD target tissue. Target tissue (tongue) from animals with acute (n = 3) and chronic (n = 3) SGVHD was harvested and analyzed for levels of cytokine mRNA production by quantitative PCR. Results are normalized against the mRNA transcript levels for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). ∗∗∗The mRNA transcript expression was below detectable levels.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )

6. Figure 5
Expression of cell-surface co-stimulatory ligand/receptor mRNA by CLIP-reactive T cells in acute and chronic SGVHD. Splenic lymphocytes were harvested from animals with early-onset acute (day 7) and chronic (day 42) SGVHD. The cells were left unfractionated or were separated flow cytometrically on the basis of reactivity with the soluble MHC class II immunoglobulin construct loaded with the N- or C-terminal CLIP variants. Preferential N- or C-terminal flanking domain reactivity was confirmed by assessing the ability to kill target cells loaded with the truncated CLIP variants. The cells were assessed for B7.1, B7.2, CD28, and CTLA-4 mRNA transcript levels by quantitative PCR. The results were normalized against levels of GAPDH mRNA transcripts. Identical results were obtained by analyzing cells from animals with acute and chronic SGVHD from 3 separate experiments. GAPDH indicates glyceraldehyde-3-phosphate dehydrogenase.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )

7. Figure 6
Functional and phenotypic analysis of N- and C-terminal CLIP-reactive T lymphocytes in acute and chronic SGVHD. T-lymphocyte clones established from animals with SGHVD were assessed for their ability to kill target cells loaded with the truncated CLIP variants at a 15:1 effector-target cell ratio; results are expressed as the percentage of specific killing ± SD. Quantitative PCR was used to evaluate the clones for cytokine (IFN-γ and IL-10) and cell-surface receptor/ligand (CD28, B7.1, and B7.2) mRNA transcripts. Results were normalized against glyceraldehyde-3-phosphate dehydrogenase mRNA transcript levels. BD indicates binding domain. ∗∗No mRNA transcripts were detected.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )