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Blood, Vol. 105, Issue 8, 3238-3246, April 15, 2005
Physiological relevance of antigen presentasome (APS), an acquired MHC/costimulatory complex, in the sustained activation of CD4+ T cells in the absence of APCs
Blood Zhou et al.
105: 3238
Supplemental materials for: Zhou et al, Vol 105, Issue 8, 3238-3246
Files in this Data Supplement:
- Figure S1. CD4+CD80acq T cells do not upregulate CD80 mRNA, but acquire CD80 and IEk and are free of contaminating APCs (PDF, 90 KB) -
(A) CD80 mRNA expression in CD4+CD80acq T cells was analyzed using RT-PCR. Lane A shows a 517–base pair (bp) fragment of CD80 cDNA amplified by RT-PCR, DNA-molecular size standard; lane B, fibroblast transfected with plasmid expressing CD80 (positive control); lanes C and D, 2 different preparations from naive (unstimulated) PCC/TCR-Tg CD4+ cells; lane E, PCC/TCR-Tg CD4+ cells stimulated for 24 hours in the presence of APCs (fibroblasts expressing CD80) and 10 µg/mL of PCC peptide that had acquired CD80 (Figure 4D). These cells were depleted of APCs as described in "Materials and methods." The RT-PCR data were published by Sabzevari et al.1 (B) Naive CD4 T cells were cocultured with P13.9 APCs pulsed with 10 µg/mL PCC peptide for 20 hours. Upon separation from the APCs, the acquisition of CD80 and IEk on the CD4+CD80acq T cells was analyzed by FACS. The results shown here are representative of the results of at least 3 separate experiments.
- Figure S2. Separation of CD4+/CD80acq T cells from APCs (PDF, 41 KB) -
Negatively selected naive PCC TCR-Tg CD4+ T cells were incubated with APCs (P13.9 or DCEK fibroblasts pulsed with 10 µg/mL PCC peptide for 3 days) for 20 hours. To remove all APCs, fibroblasts were fed with Dynal beads to enable us to separate them magnetically from the T cells. The obtained CD4+CD80acq T cells were free of contaminating APCs (98-99% purity, Figure S1B).
- Figure S3. Acquisition of CD80 and IEk by CD4 T cells from APCs expressing high or low levels of CD80 (P13.9 and DCEK as APCs) (PDF, 69 KB) -
(A) P13.9 and DCEK cell lines were derived from the same fibroblast cell line, and were stably transfected with IEk and CD80 expression vectors. Both P13.9 and DCEK APCs expressed similar levels of MHC II (IEk); P13.9 APCs expressed a higher level of CD80, whereas DCEK APCs expressed a much lower level of CD80 (filled peak represents the anti-CD80 isotype control; outlined peak is anti-CD80 Ab). These cell lines were checked by 2-color FACS analysis. (B) Acquisition of CD80 by CD4+ T cells from APCs that express high level of CD80 (P13.9) or low level of CD80 (DCEK). These results are representative of at least 3 separate experiments.
- Figure S4. Activation and proliferation of CD4+CD80acq T cells by DCEK (APCs expressing low levels of CD80) mixed with different proportions of P13.9 (APCs expressing high levels of CD80) (PDF, 215 KB) -
(A) Naive CD4+ T cells were cocultured with PCC-pulsed DCEK mixed with graded amount of PCC-pulsed P13.9 for 20 hours. After the separation from APCs, levels of CD80, CD25, and CD69 on CD4+CD80acq T cells were analyzed by FACS. These results are representative of 2 separate experiments. (B) Proliferative response of these CD4+CD80acq T cells after the separation from APCs was measured using 3H uptake assay. Error bars indicate standard deviations based on the mean of triplicate wells on 96-well culture plates. These experiments were repeated twice.
- Figure S5. Normalization of bands in RNase protection assay (PDF, 38 KB) -
The band densities of the cytokines in RNase protection assays (Figure 3A in text) were normalized according to the band density of GAPDH (housekeeping gene) using the Scion Image analysis system (Scion, Frederick, MD). Cytokine expression in CD4+CD80acq hi T cells was examined using RPA when these T cells were incubated in the absence or presence of 1 µg/mL PCC without APCs at various time points.
- Figure S6. Thioglycollated-elicited macrophages express CD80 and IEk and can serve as APCs (PDF, 28 KB) -
PCC/TCR-Tg mice were injected intraperitoneally with 0.8 mL 4% thioglycollate. Four days later, cells from peritoneal exudates were harvested and cultured for 3 hours in complete media. The nonadherent cells were removed by several washes, and adherent cells were further cultured in the presence of 10 ng/mL IL-4 for 2 days and then analyzed by FACS. As shown in this figure, all adherent cells were macrophages (ie, expressed CD11b/Mac-1) and were positive for CD80 and IEk (the filled peak represents the isotype control Ab). These results are representative of 2 separate experiments.
- Figure S7. CD4+ T cells acquire CD80 and IEk from macrophages; the resulting CD4+CD80acq T cells are capable of proliferating in the absence of APCs/macrophages (PDF, 50 KB) -
CD4+ T cells were cocultured with PCC-pulsed macrophages (APCs were pulsed with 10 µg/mL PCC peptide) for 20 hours. After the separation from macrophages, CD4 T cells that had acquired CD80 and IEk — CD4+CD80acq T cells — were further treated with 0.1% pronase for either 12 or 30 minutes. Levels of CD80 and IEk were analyzed by FACS before and after the treatment with pronase. After pronase treatment, T cells were cultured for an additional 24 hours in the absence of APCs or peptide, and their proliferative response was measured using 3H uptake assay. Error bars indicate standard deviations based on the mean of triplicate wells on 96-well culture plates. These experiments were repeated twice. Sabzevari H, Kantor J, Jaigirdar A, et al. Acquisition of CD80 (B7-1) by T cells. J Immunol. 2001;166: 2505-2513.
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