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Blood, Vol. 113, Issue 22, 5516-5525, May 28, 2009
Reciprocal responsiveness to interleukin-12 and interferon- specifies human CD8+ effector versus central memory T-cell fates
Blood Ramos et al.
113: 5516
Supplemental materials for: Ramos et al
Files in this Data Supplement:
- Figure S1. IL-12– and IFN-α–regulated Perforin and Granzyme B expression (JPG, 259 KB)
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CD8+ CD45RA+ were purified from peripheral blood and cultured in the presence or absence of polarizing cytokines for 7 days. (A) Rested cells were harvested and stained for intracellular content of perforin and granzyme B in 3 separate donors and assessed for expression by flow cytometry. (B) Quantification of mean fluorescence intensities for perforin (top panels) or granzyme B (bottom panels) in three separate human donors.
- Figure S2. IL-12–driven CTL activity is perforin-dependent (JPG, 14.9 KB)
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Day 7, IL-12–polarized cells were used as effectors in a 51Cr re-directed lysis assay. THP-1 target cells were prepared as described in figure 1 and effector cells were either left untreated (open circle) or treated with concanamycin A (1µM) for 90 minutes (open triangle). As a control for the vehicle, effectors were treated in 1µM DMSO (open square). Cells were incubated with target cells at the indicated effector:target ratios for 4hrs and CTL activity was assessed by quantification of release of 51Cr into the supernatant by β emission counting.
- Figure S3. T-bet expression correlates with the TEM phenotype in human CD8+ T cells (JPG, 156 KB)
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Examination of T-bet expression as a function of cytokine, chemokine receptor and lytic effector molecule expression. (A) Day 7 IL-12 + IFN-α polarized Cells were activated for 4hrs with PMA and Ionomycin in the presence of brefeldin A, and live CD8+ gated cells were examined for IL-2 and IFN-γ expression by bi-variant dot plot analysis. Cells were gated as shown and assessed for T-bet expression as a function of cytokine expression. (B and C) Resting CD8+ cells were gated based on expression of CCR7 and CXCR3 (B) and perforin and granzyme (C) and expression of T-bet was assessed by intracellular flow cytometry.
- Figure S4. CCR7hi/CXCR3lo sorted cells maintain viability following restimulation (JPG, 173 KB)
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CD8+ CD45RA+ cells were polarized to day 7 with IL-12 + IFN-α and sorted into either CCR7hi/CXCR3lo or CXCR3hi/CCR7lo populations. Sorted cells were either left untreated (resting) or activated on 1.5µg/ml anti-CD3–coated plates for 3 days. At day 3, CCR7hi sorted (left panel) or CXCR3hi sorted (right panel) cell survival was assessed by forward and side scatter bi-variant dot plot profiles (A), and by 7-amino-actinomycin D staining (B).
- Figure S5. CCR7hi/CXCR3lo TCM cells display functional memory responses and are distinct from naïve cells (JPG, 199 KB)
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CD8+ CD45RA+ cells were cultured to day 7 with IL-12 + IFN-α either in the absence (Naïve) or presence of anti-CD3/anti-CD28 (Memory). CCR7hi/CXCR3lo cells were then sorted from these cultures (1° Activation, top panels), labeled with PBSE, and then either left untreated (resting), or restimulated with 1.5µg/ml anti-hCD3 for 24hrs. Cells were assessed for forward and side scatter profile (2° Activation, top panel), proliferation by PBSE dilution (2° Activation, middle panel) or granzyme B as a function of division (2° Activation, bottom panel).
- Figure S6. Reciprocal regulation of the IL-12Rβ2 and IFNAR2 on developing TEM and TCM cells (JPG, 118 KB)
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CD8+ CD45RA+ sorted cells were labeled with CFSE and cultured in the presence or absence of cytokines for 3 days. (A) Examination of expression of IL-12Rβ2 and IFNAR2 by division on day 3 of activation. Top: division of total live CD8+ population; middle: IL-12Rβ2 expression; bottom: IFNAR2 expression. (B) Quantification of mean fluorescence intensities as a function of division for IL-12Rβ2 (top panel) or IFNAR2 (bottom panel). Black, open square (neutralized), magenta, closed circle (IL-12), teal, open circle (IFN-α), orange, closed square (IL-12 + IFN-α).
- Figure S7. Development of TEM and TCM populations of human CD8+ T cells is directly affected by the concentration of IL-12 and IFN-α (JPG, 172 KB)
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CD8+ CD45RA+ cells were purified, labeled with CFSE, and cultured to day 7 under polarizing cytokine conditions. Cells were either left untreated (Neut.), treated in the presence of IFN-α alone (1000u/ml) or treated with IL-12 and a titration of IFN-α at 0u/ml, 10u/ml, 100u/ml, 1000u/ml or 5000u/ml. (A) Rested cells were harvested and assessed for division by CFSE dilution (left panel) or development of CCR7hi TCM and CXCR3hi TEM (right panel) either in the presence of IL-12 alone (top panel) IFN-α alone (middle panel) or IL-12 + IFN-α (bottom panel). (B) Quantification of the percent of live cells falling within either the division 0 (left panel, magenta), divisions 1–3 (middle panel, teal) or divisions 4+ (right panel, orange) as a function of primary cytokine polarizing conditions.
- Figure S8. IL-12 and IFN-α/β direct the development of effector and central memory populations of human CD8+ T cells (JPG, 141 KB)
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Naïve CD8+ CD45RA+ T cells receiving signals 1 and 2 require further cytokine signals to develop into effector and memory populations. IL-12, programs the development of rapidly dividing cells, which acquire full effector properties. Alternatively, cells responding to IFN-α/β do not divide rapidly to primary activation but rather acquire phenotypic and functional attributes of TCM. Programming of TEM and TCM occurs through the differential expression of the IL-12R and the IFNAR and directly correlates to differential downstream responsiveness to cytokine signaling. The pathways to TCM and TEM are further modulated by the strength of initial TCR activation. Under conditions of strong TCR activation, cells progress toward the TEM fate, whereas weaker signal strength dampens this progression and favors a balance between TCM and TEM. Taken together, these results suggest a model in which development of TCM and TEM phenotypes occurs through signals obtained through both the TCR and cytokine signaling pathways. Thus, the development of TCM and TEM during infection may be the direct result of the magnitude of inflammatory cytokine signals received by developing T cells at the time of priming.
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