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. Author manuscript; available in PMC: 2010 Nov 15.
Published in final edited form as: J Immunol. 2009 Oct 19;183(10):6346–6358. doi: 10.4049/jimmunol.0901773

Figure 1.

Figure 1

Autologous hematopoietic stem cell transplantation (Post-t) reduced IFN-γ, but increased IL-13 responses of CD4+ T cell in fresh PBMC to nuclesomal autoepitopes. A, Fresh PBMC samples were cultured with nucleosomal histone peptide epitopes, or whole nuclesomes (Nuc.) in the presence of IL-2, IL-7, IL-15 and anti-CD28/CD49d for 3 days, and then stained for surface CD4 and intracellular IFN-γ or IL-13 for flow cytometry analysis of gated CD4+ T cells. Peptide epitope designations are abbreviated to fit X-axis, for example H1′22-42 is H1, H3115-135 is H3(115), and so on. The baseline values of IFN-γ response of CD4 T in PBMC (cultured in medium only) were 0.06-0.27, and the baseline values of IL-13 response of CD4 T (medium only) were 0.07-0.34. Cytokine Response Index (CRI) ratios were calculated by dividing experimental values by corresponding values of resting control CD4 cells (without autoantigen stimulation). CRI below 2 is considered to be at background. The horizontal line indicates the mean. We used a BRN-3 transcription factor related peptide, called Eluted Peptide-2 (EP-2) (sequence DWMEEEEGAQREKE) as a negative control peptide for the histone peptide epitopes (8) and OVA323-339 as an irrelevant control antigen for nucleosomes; and the positive control stimulation was done with anti-CD3. The CRI for IFN-γ response of CD4 T cells to EP-2 were 0.5-1.6, to OVA were 0.4-1.2, and to anti-CD3 were 12.7-51. The CRI for IL-13 response of CD4 T cells to EP-2 were 0.4-1.8, to OVA were 0.6-1.5, and to anti-CD3 were 9.3-46. B, Post-transplant PBMC were depleted of CD8 T cells and then cultured with nucleosomal autoepitopes to measure IFN-γ response of CD4 T cells as in panel A, along with two other groups: whole PBMC group and a group with CD8 T cells added back to PBMC which were depleted of CD8 T cells. Experiments were repeated five times and the bars show the mean ± SD. * p < 0.01. C, CD4+CD25high T cells or CD8 T cells were removed from post-transplant PBMC by sorting, then the whole PBMC, or PBMC without CD4+CD25high or without CD8 T cells were cultured with nucleosomal autoepitopes to measure IFN-γ response of CD4 T cells as we did in panel A. Experiments were repeated three times and bars show the mean ± SD.