Human Correlation Experimental Data

Introduction The overall workflow for experiments using primary human cells is shown in Figure 1. Blood is collected from normal human donors, anticoagulated with ACD, and lymphocyte subsets are purified using magnetic beads. Gene knock down approaches used by the core are performed using commercially available reagents and include for example, Accell siRNA (Dharmacon), Nucleofection (Lonza / Amaxa) and Lentiviral transduction and expression of shRNA (Openbiosystems). Before addition of lentivirus the cells are stimulated using immobilized anti-CD3 / anti-CD28 in addition to having IL-2 present in the culture medium. Transduced cells are selected using puromycin and verified by expression of GFP and specific knock down of targeted genes using qRT-PCR. 

Transient RNA knock-down using nucleofection or direct uptake of siRNA into cells Examples of nucleofection and direct uptake of siRNA into PBMC are shown in Figures 2 and 3. Nucleofection enables efficient uptake of plasmid DNA as large as the pGIPZ transfer vector into PBMC with expression of GFP within 24 hours. Accell siRNA (Dharmacon) added to PBMC at micromolar concentration (without transfection reagent) is taken up by cells as shown by FAM fluorescence of the labeled siRNA and knock-down of specific RNA (Figure 3).

Lentiviral transduction of proliferating lymphocytes When PBMC or purified CD4 or CD8 lymphocytes are stimulated with immobilized anti-CD3 / anti-CD28 and IL-2, cell aggregates began to form within a few hours, and when Lentivirus is added substantial numbers of cells are transduced and can be observe to express GFP by fluorescence microscopy within several days (Figure 4). Puromycin (4 µg/ml) can be used to eliminate non-transduced cells and is best added within 24 hours of transduction. Figure 4 shows prominent GFP expression in transduced cells that were maintained in culture for 6 days without puromycin.

Knock-down of hnRNPLL using lentiviral expression of siRNA One of the genes shown in the forward genetics studies to be important for adaptive immune responses is the heterogeneous nuclear ribonucleoprotein, hnRNPLL, which is required for alternative splicing of CD45. HnRNPLL is a regulated gene product in lymphocytes, with the lowest expression in B cells which express B220 / full length CD45ABC compared to high expression in memory T cells which express CD45RO. The ENU mutant mouse, Thunder (Th-under) with a V136D substitution in hnrnpll was found to be unable to silence exons 4, 5 and 6 of CD45 (Wu, et al. Immunity 29:863, 2008). CD45 exon silencing was restored in thu/thu T cells that were transduced with wild-type hnrpll cDNA. In other studies, an shRNA screen identified hnRNPLL as necessary and sufficient for signal-induced exon skipping of CD45 (Oberdoerffer, et al, Science 321:686, 2008).

Knock-down of hnRNPLL in primary CD4 lymphocytes was performed using specific lentiviral transfer vector from OpenByosystems (Figures 6-8 and Table I). Blood was collected from a normal human donor using ACD anticoagulant followed by purification of PBMC using Ficoll-Paque and direct immunomagnetic separation of CD4 cells using Miltenyi reagents. The purified CD4 cells were cultured under conditions of costimulation with immobilized anti-CD3/anti-CD28 and stimulation with IL-2 (20 U/ml). After four hours, packaged lentivirus (culture supernatant from HEK293 cells) was added to the proliferating PBMC followed by overnight incubation at 37°C with 10% CO₂. Puromycin, 4 μg/ml final, was added 24 hours after transduction, and the cells were maintained in culture for several weeks, removing spent medium and replenishing with DMEM + 10% FBS containing Puromycin at 3-5 day intervals. After three weeks in culture the cells were essentially 100% GFP positive, and qRT-PCR demonstrated substantial knock-down of hnrpll message (Figure 6). CD4+ cells transduced with non-targeting shRNA, empty cloning vector or two shRNA vectors specific for hnrpll were stimulated for three days with IL-2, IL-2 + PMA or IL-2 + PMA + Ionomycin followed by antibody staining and analysis using an LSRII flow cytometer. As shown in Figure 7, amine-reactive fixable viability stain was used to select viable cells followed by gating on GFP-expressing cells which were observed to be essentially 100% CD3+, CD4+. Knock-down of hnRNPLL using the 301 vector reduced expression of CDRO, and the effect was even more substantial when the 303 vector was used (Figure 8, Table I).

The tabulated data from flow cytometric analysis (Figure 8) demonstrate that shRNA induced knock down of hnrnpll substantially reduced expression of CD45RO in cells expressing the vectors 301 and 303 compared with cells transduced with non-targeting or empty cloning vector.

* Percent of total