To extend our experiments from looking at the amounts of B virus produced from HFFs during co-culture, we wanted to determine how different doses of B virus infected cell lysates would affect IFN-
production from pDCs. We chose IFN- as the measure of the pDC response to B virus infected cell lysates because pDCs are the most potent producers of Type I IFNs in response to viral nucleic acids. Our previous experiment showed that the titers pDCs were exposed to in co-culture with B virus infected
HFFs were about 4 times lower than the titers of B virus released from infected cell lysates. Based on these data we hypothesized that pDC-induced IFN- correlated directly with virus dose to which these
were exposed. To determine the effect of virus dose on the ability of pDCs to produce IFN-, freshly iso-
lated pDCs from pooled PBMCs collected from seronegative donors were exposed to B virus at 2, 5, 10 and 20 pfu/cell. To ensure that the pDCs were responsive to B virus two positive controls were used, ie., HSV-1 at 5 pfu/cell and CpG 2216 at 5g/ml. The pDCs were seeded in 50ul of culture medium into 96- well round-bottomed plates and each treatment was added in 150ul of culture medium. pDCs were cul- tured at 37C, in a 5% CO2 humidified incubator,and at 24h cell-free supernatants were centrifuged at
100xg for 10min to remove cells and debris and tween/DOC was added to a final concentration of 1% to inactivate virus. Supernatants were stored at -80C until analyzed by ELISA for IFN- using the Human IFN- pan ELISA Development Kit (HRP) (MabTech, Cincinnati, OH). This kit detects 11 of the 12 subtypes of IFN- using a sandwich ELISA technique with a capture antibody and a detection antibody conjugated with horseradish peroxidase (HRP). Serial dilutions of a standard provided with the kit allowed for gen- eration of a standard curve and unknown IFN- concentrations were interpolated using RIA
Spline/LOWESS in GraphPad PrismTM. As shown in Figure 19, the pDCs responded to B virus released from the infected cells during the course of infection with the production of IFN- although it was in- versely correlated to the dose of B virus. Both of our positive controls verified that the pDCs were re- sponsive by producing IFN- in response to HSV-1 infected cell lysates and CpG 2216 with 33 fg/cell and 42 fg/cell, respectively. The pDCs exposed to B virus released from infected cell lysates at 2 pfu/cell (or 2x104 pfu) produced 33.5 fg/cell of IFN-, while pDCs exposed to B virus released from infected cell ly- sates at 5 pfu/cell (6x104 pfu) produced 28 fg/cell of IFN-, while high MOIs of B virus released from in- fected cell lysates at 10 pfu/cell (1.2x105 pfu) and 20 pfu/cell (2.4x105 pfu) failed to induce significant amounts of IFN- at 4 and 5 fg/cell, respectively. Neither of the two negative controls, cells exposed to medium or MCL (at the protein equivalent to BV 20 pfu/cell), produced any IFN-, indicating that the
IFN- produced was a specific response to virus. This result was unexpected because independent stud- ies with HSV-1 have shown that the more virus, the greater the amount of IFN- pDCs produce, indicat- ing that high MOIs of B virus may high MOIs of block or overwhelm pDC IFN- production. These data support our hypothesis that IFN- production by B virus exposed pDCs correlates inversely with the amount of virus used to expose pDCs, surprisingly. Based on these data and the data shown in Figure 18, the pDCs in co-cultures with B virus infected HFFs (1.8x104 pfu) should produce similar amounts of IFN- as pDCs in direct exposure to B virus infected cell lysates (5x104 pfu) as there was no significant differ- ence of IFN- produced from pDCs exposed to 2 or 5 pfu of B virus, both of which had significantly high- er production of IFN- than pDCs exposed to MCL (Figure 19). To explain the drastic reduction of IFN- production from pDCs in co-cultures when compared to pDCs directly exposed to B virus infected cell lysates, we considered the quality of virus within the virus lysates as these virions are likely surrounded by the nuclear or Golgi membranes whereas the virions released from infected cells in co-cultures would be surrounded by plasma membranes acquired when virions egress out of infected cells by exocytosis. Another possible explanation for the increase of IFN- in pDCs directly exposed to B virus infected cell lysates is that these contain free viral DNA that is available for the pDCs to endocytose and activate TLR9 dependent IFN- production. There is likely to be very little free DNA in the co-culture system, as only intact virions would be released with the DNA protected within the enveloped capsid. Future studies are needed to fully understand the mechanism of a differential response between B virus infected cell lysates and the B virus virions produced from infected HFFs in co-cultures.
Figure 19 IFN- production from pDCs is inversely correlated to virus within infected cell ly- sates
pDCs were isolated by negative selection from pooled, fresh PBMCs obtained from seronegative donors and were distrib- uted equally into wells of a 96-well plate. pDCs were exposed to B virus in infected cell lysates with titers ranging from 2- 20 pfu/ml, as determined by standard plaque assay. pDCs were cultured for 24h and cell-free supernatants were collected and tween/DOC, added to a final concentration of 1%. IFN- levels were determined using ELISA. As negative controls, medium treated pDCs and MCL treated pDCs, at a level equivalent to BV 20 pfu, were used. As positive controls, pDCs were exposed to HSV-1 at 5 pfu/cell and pDCs were exposed to 10ug/ml of CpG 2216. **p<0.05, NS=not significant. ANOVA was used to analyze results along with Newman-Keuls post hoc test for pair comparisons.