A titration of the amount of interferon-a required to activate NK cells

The results in Section 4.2.11 showed that the activation of NK cells by 1000 IU/ml of interferon-a reached a maximum after 4 hr of treatment. It was also shown that the activation of NK cells by CMV required between 6 and 20 hr of activation, during which times 40-303 IU/ml of interferon-a was produced. In order to ascertain whether the latter amounts of interferon-a were in fact sufficient to activate NK cells within 4 hr, the effect of treating NK cells with similar concentrations of interferon-a to those measured in Section 4.2.11 was examined. PBMCs were treated for 4 hr with medium alone or a range of concentrations of interferon-a from 0 to 1000 IU/ml. The cells were then washed, and tested in a 4 hr cytotoxicity assay at an effector to target cell ratio of 50:1 for their ability to lyse CMV-infected target cells. The target cells were fibroblasts that had been infected with CMV strain AD 169 at an MOI of 2 for 4 days.

The results are shown in Figure 4-16, where the extent of activation of the NK effector cells was assessed by their ability to lyse target cells within a 4 hr cytotoxicity assay. During the 4 hr period of incubation with interferon-a, NK cells were partially activated by amounts of interferon-a as low as 4 IU/ml. The extent of NK cell activation as measured by their lysis of target cells increased with increasing concentrations of interferon-a up to 111 IU/ml. Thereafter, higher concentrations of interferon-a did not result in increased NK cell activation. From these results it could be concluded that the amounts of interferon-a produced after the incubation of PBMCs with cell-free CMV were sufficient to activate NK effector cells within the PBMCs. Furthermore, the amounts of interferon-a produced at the various time points in Figure 4-15 were consistent with the extent of NK cell activation observed at these time points. For example, the amount of interferon-a produced by the incubation of PBMCs with cell-free CMV for 4 hr was 11.5 IU/ml, which, based on the results shown in Figure 4-16 was sufficient to cause partial activation of NK cells. After 6 hr, 40 IU/ml of interferon-a was produced, which activated NK cells to a similar extent to that shown by 37 IU/ml of interferon-a in Figure 4-16. Finally,

after 20 hr, the 303 IU/ml of interferon-a produced was sufficient to cause maximal activation of NK cells, as shown by the addition of 333 IU/ml in Figure 4-16.

The activation of NK cells was shown in the previous section to occur rapidly in response to interferon-a. Thus, it appears that the extended time taken for the activation of NK cells by cell-free CMV is probably required largely for the production of interferon-a at sufficient levels to activate NK cells. The time required for the production of such levels of interferon-a by PBMCs in response to cell-free CMV, was between 6 and 20 hr. Then, the interferon-a produced by PBMCs in response to cell-free CMV could rapidly activate NK cells such that they were capable of lysing target cells in a 4 hr cytotoxicity assay. It was shown here that the amounts of interferon-a produced by PBMCs after various times of exposure to cell- free CMV as shown in the previous section, were sufficient to activate NK cells to a similar extent as was observed at those various times.

(/) (/) 30- 20^ 10- 0 4 12 37 111 333 1000 IFN-a (lU/m I)

Figure 4-16 A titration of the amount of interferon-a required to activate NK ceils

PBMCs were isolated, and then incubated for 4 hr with various amounts of interferon-a as shown on the x-axis. The cells were then washed, and used as effector cells in a 4 hr cytotoxicity assay. The target cells were fibroblasts that had been infected with CMV strain AD 169 at an MOI of 2 for 4 days. The results are expressed as the percentage (%) lysis at an effector to target cell ratio of 50:1, and represent the mean ± standard error of triplicate values.

4.2.13 The measurement of IL-2, lL-12, IL-18, TNF-a and interferon-/ produced by PBMC after incubation with cell-free CMV

The above results showed that interferon-a was produced by PBMCs in response to their incubation with cell-free CMV, and that it could activate NK cells. This strongly suggested that interferon-a was the substance produced by DR+ cells in response to CMV that then activated NK cells. However, it was possible that other cytokines could contribute to the activation of NK cells by CMV. Thus, cell culture supernatants from PBMCs that had been activated overnight with cell-free CMV, were tested by immunoassay for the presence of cytokines known to activate NK cells. PBMCs were incubated overnight with 6x10® pfu/ml of cell-free CMV strain A D I69 or medium alone as a control. The culture medium was then harvested, clarified by centrifugation and immediately tested by immunoassay for the presence of IL-2, IL-12, IL-18, TNF-a and interferon-y.

The results are shown in Table 4-1. In contrast to the case for interferon-a, no IL-2, IL-12, IL-18, TNF-a or interferon-y was detectable by immunoassay in any of the supernatants tested. The lower limits of detection of the various immunoassays were well below the levels required for the activation of NK cells by these cytokines. These results indicated that none of these cytokines were produced after the incubation of PBMCs with cell-free CMV, and therefore that they probably did not contribute towards the activation of NK cells under these conditions.

Table 4-1 The production of cytokines by PBMCs activated by incubation with cell-free CMV

Cytokine Level (pg/ml)^ Sensitivity^

IL-2 <7 pg/ml 7 pg/ml

IL-12 <0.5 pg/ml 0.5 pg/ml

IL-18 <15 pg/ml 15 pg/ml

Interferon-y <3 pg/ml 3 pg/ml

TNF-a <4.4 pg/ml 4.4 pg/ml

PBMCs were incubated for 24 hr with 6 x 1 0 ® pfu/ml of CMV strain AD169. The culture supernatant was harvested, clarified by centrifugation and then assayed for the presence of cytokines

4.2.14 A comparison of the phenotype of CD56+ effector cells