Cálculo del volumen del reactor de tipo flujo pistón

Figure 4-18 Interferon-a-mediated protection of uninfected fibroblasts from NK cell-mediated lysis

Fibroblasts were left uninfected (UNINF), or were infected with the AD169 strain of CMV at an MOI of 2 (AD169). 4 days after infection, the cells were radiolabelled, and either treated with 1000 lU/ml interferon-a (+IFN-a) overnight or left untreated (- IFN-a). PBMCs were incubated with medium alone as a negative control (CONTROL) or were activated with 1000 lU/ml interferon-a overnight, and then used as effector cells in a 4 hr cytotoxicity assay against the various fibroblast target cells as shown. 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.

4.3

Discussion

In Chapter 3, the requirements for measuring the lysis of CMV-infected target cells were investigated. These experiments revealed that the 20 hr cytotoxicity assay for the measurement of NK cell-mediated lysis of CMV-infected cells could be separated into an overnight activation phase and a 4 hr effector phase. The activation phase depended on a soluble factor released from CMV-infected cells. The aim of the experiments in the present chapter was to examine further this phase involving the activation of NK cells by CMV-infected cells. Initially, in this chapter it was shown that NK cells could also be activated by cell-free CMV viral inoculum, and that the presence of infected cells was not necessary. Thereafter, all the experiments were performed using cell-free CMV, rather than infected cells as in the previous chapter. Thus, NK cells were activated overnight, using cell-free CMV, after which, they could lyse infected or uninfected target cells in a 4 hr cytotoxicity assay. In the absence of prior activation, NK cells were unable to lyse target cells in the 4 hr cytotoxicity assay.

The investigation of the activation of NK cells by CMV required the identification of the virus/virally-induced factor produced by CMV-infected cells, that was responsible for the ultimate activation of NK cells. In Chapter 3, this had been found to be a soluble factor that was produced late in infection. There were two possibilities that were investigated, which were not mutually exclusive. The first, that the cell-free CMV viral inoculum produced by infected cells contained cytokines that were capable of activating NK cells either directly or indirectly. Secondly, that virus particles produced late in the viral life cycle either indirectly or directly, could activate NK cells. In order to answer these questions, cell-free CMV strain AD 169 virus inoculum was fractionated by ultracentrifugation, into a virus-free supernatant, and purified virus particles. The testing of these two fractions revealed that all of the activatory potential of the unfractionated viral inoculum could be attributed to the purified virus particles. Furthermore, the virus particles were not required to be infectious in order for the activation of NK cells to occur, since purified virus that had been inactivated by heat or ultraviolet irradiation was still capable of activating NK cells. The retained activatory capacity of the treated virus particles was not due to

their incomplete inactivation, since no CMV immediate early antigen could be detected after the inoculation of the inactivated virus preparations onto fibroblast monolayers. These results suggested that NK cell activation by CMV did not involve infection per se, but could involve either the uptake of non-infectious virus particles, or the cell surface interaction of virus particles with NK cells or accessory cells. Whilst other studies have demonstrated the lysis of CMV-infected fibroblasts by NK cells (415,434), these studies used a 20 hr cytotoxicity assay, and did not examine the activation of NK cells by cell-free CMV. Thus, the present study was the first to show that the activation of NK cells by CMV depended on the presence of viral particles.

In order to demonstrate that such an initial interaction of cell-free CMV virions with PBMCs was required for the activation of NK cells, the effect of heparin treatment was examined. Exogenous heparin is known to prevent the binding and entry of CMV into cells, as it competes for the initial binding of the virus to the cell surface (47). The pre-treatment of virus particles with 50 lU/ml of heparin was found to significantly (p <0.01) reduce NK cell activation, as measured by the ability of NK cells to lyse both infected and uninfected target cells in a 4 hr cytotoxicity assay. The use of heparin at the higher concentrations of 100 and 200 ID/ml resulted in further inhibition of NK cell activation, indicating a dose-dependent inhibitory effect of heparin on the NK cell activation by CMV. These results suggested that virus attachment/entry into cells within the PBMC population was required for the ultimate activation of NK cells by cell-free CMV. The inhibition of NK cell activation by heparin was accompanied by a decline in the infectious potential of the virus, with virtually no infection of inoculated fibroblasts occurring after the treatment of virus with 200 lU/ml of heparin. It was shown earlier in this chapter however, that the infectivity of virus particles was not required for their ability to activate NK cells. Furthermore, in vitro generated stocks of cell-free CMV inoculum are known to contain a variable proportion of enveloped, non-infectious viral particles (4), which could still possess the ability to activate NK cells. Thus, the drawing of a parallel between the infectious capacity of virus and its ability to activate NK cells may not be applicable here. Nevertheless, the testing of virus infectivity after heparin treatment provided at least a measure of the efficacy of the treatment in preventing

virus attachment to cells, albeit to a different cell type. It was possible that the heparin treatment could have exerted a direct effect on NK cell activity, which could have accounted in part, for the effects attributed to the inhibition of virus attachment. Since it was difficult to control for this possibility, the heparin treated virus was incubated with PBMGs for only 1 hr, before washing and further incubation. There are conflicting reports in the literature on the effects of heparin on NK cell activity in vitro (435,436) and in vivo (437). In a study that showed inhibition

in vitro of NK cell activity by heparin, the heparin was present during the cytotoxicity assay (435). In the present study however, the heparin was only in contact with the PBMCs for a period of 1 hr after which it was removed, prior to overnight incubation and the cytotoxicity assay. Furthermore, similar preservative-free heparin preparations (15 lU/ml) were used as an anticoagulant for the collection of blood samples throughout this study with no apparent inhibitory effects on subsequent NK cell function.

In order to determine the amount of cell-free CMV that was required to activate the NK cells within a given number of PBMCs, the latter were treated with serial dilutions of cell-free virus. In parallel, the ability of the various dilutions of virus to infect fibroblasts was examined. These data then allowed for the calculation of the number of pfu of virus required for the optimal activation of the NK cells within 5 x 10® PBMCs, in a volume of 1 ml. The ability of cell-free virus to activate NK cells fell sharply within a range of 32 to 64-fold dilution, which translated to between 10,000 and 20,000 plaque forming units of virus that were required for the activation of NK effector cells within 5x10® PBMCs. Thus, it appeared that only a relatively low ratio of plaque forming units of virus to PBMCs was required for the activation of NK cells. However, these calculations were based on the number of infectious virus particles, and as mentioned earlier, the cell-free virus used was likely to also contain non-infectious particles. Unfortunately, it was not possible to measure the total number of both infectious and non-infectious virus particles in the cell-free virus preparation. Nevertheless, these results indicated that the activation of NK cells by cell-free CMV was dependent on the dose of virus used, and provided a relative measure of the amount required for future experiments.

In Chapter 3, a period of 16-20 hr had been found to be sufficient for the activation of NK cells by CMV-infected cells to occur. In the present chapter, using cell-free CMV, the activation of NK cells also occurred within 16-20 hr. It was possible however, that the activation of NK cells by cell-free CMV could be achieved in a shorter period, and therefore the activation of NK cells after their incubation with cell-free CMV for various times was measured. NK cell activation was first apparent after 4 hr of incubation with cell-free CMV, increasing up to the longest time tested of 20 hr. 6 hr of incubation with cell-free CMV resulted in almost as much activation as 20 hr, suggesting that a time point between 6 hr and 20h might have yielded the maximal level of NK cell activation. However, an overnight activation time of 16-20 hr was the most logistically convenient, and so this was the time used for future experiments. NK cells, which form part of the innate immune system, are not MHC- restricted and do not depend on immunological memory for their effector functions. Indeed, in previous studies by others, the NK cells from either seropositive or seronegative donors could mediate the lysis of CMV-infected cells in a 20 hr cytotoxicity assay (415,434). Nevertheless, in this study, the possibility that the time taken to activate NK cells by cell-free CMV might differ between the two types of donor was considered. This was thought to be relevant, since it was possible, if unlikely during the short times used here, that pre-existing immunity to CMV could augment NK cell-activation via the induction of cytokines by memory cells. However, no differences were observed in the time taken for cell-free CMV to activate NK cells from either seronegative or seropositive donors.

Next, the cell types involved in the activation of NK cells by CMV were investigated. In Chapter 3, it was shown that if PBMCs were depleted of CD56+ cells, they were unable to lyse CMV-infected target cells in a 20 hr cytotoxicity assay. Here, PBMCs were first activated by cell-free CMV, and then sorted on the basis of their CD56 antigen expression before testing them for their cytotoxicity against infected or uninfected fibroblast target cells in a 4 hr cytotoxicity assay. The depletion of CD56+ effector cells abrogated the ability of the PBMCs to lyse target cells. This showed that once activated, CD56+ cells were the effector cells solely responsible for the lysis of CMV-infected or uninfected fibroblast target cells. In Chapter 3, however, the removal of CD56- cells from PBMCs abrogated the ability of CD56+ cells to lyse

infected target cells in the 20 hr cytotoxicity assay. Taken together with the fact that activated CD56+ cells were shown to be responsible for target cell lysis, this implied that accessory cells within the CD56- cell population were required for the activation of NK cells by cell-free CMV. Indeed, when, prior to incubation with cell-free CMV, DR+ cells were depleted from PBMCs, NK cells within the DR- cell fraction were not activated such that they could lyse target cells in a 4 hr cytotoxicity assay. Dual chamber transwell experiments showed that during the activation of NK effector cells in this system, contact between DR+ and DR- cells was not required. Taken together, these results indicated that a soluble factor produced by DR+ cells in response to cell-free CMV, was responsible for the activation of CD56+ NK effector cells in this system. Others have shown the need for DR+ accessory cells to be present in order to observe the lysis of CMV-infected target cells in a 20 hr cytotoxicity assay (418,434). In those reports, it was suggested that DR+ cells might be involved in the activation of NK cells by CMV-infected cells. However, a 20 hr cytotoxicity assay was used and hence the effects of DR+ cell depletion on the activation phase alone were not addressed, as in the present study.

In the present study, the requirement for DR+ accessory cells for the activation of NK cells by cell-free CMV could b/ bypassed by the addition of interferon-a. This suggested that interferon-a could be the soluble factor produced by DR+ cells exposed to cell-free CMV, which was responsible for the activation of NK cells. In support of interferon-a being the factor responsible for the indirect activation of NK cells by CMV, this was the only cytokine known to be able to activate NK cells, which was detected in the supernatant after exposure of PBMCs to CMV. In previous studies, interferon-a had been measured in the co-culture supernatants of CMV-infected cells and PBMCs (418). In the present study, the amount of interferon-a produced after various times of activation by cell-free CMV paralleled the level of NK cell activation after these times. Furthermore, the addition of similar levels of exogenous interferon-a to those measured in the supernatants of PBMCs incubated with cell-free CMV for various times, resulted in similar levels of activation. The findings here of the amounts of interferon-a required to activate NK cells, as well as the time required, are in broad agreement with those of others (438). However, the latter authors were studying the lysis of K562 cells, which do

not require the prior activation of NK cells, although interferon-a can augment the level of lysis. It was found that NK cell cytotoxicity towards K562 cells was augmented by amounts of interferon-a as low as 10 lU/ml, and that the induction occurred rapidly. Based on the results here, the induction by cell-free CMV, of sufficient levels of interferon-a to cause maximal stimulation of NK cells required in excess of 6 hr. Furthermore, the activation of NK cells by such concentrations of interferon-a could occur within 4 hr. Adding together these times, plus the 4 hr duration of the cytotoxicity assay, would account for the extended 20 hr cytotoxicity assay required to measure the lysis of CMV-infected fibroblasts by non-activated NK cells.

Finally, after the incubation of PBMCs with either cell-free CMV or interferon-a, the cell surface phenotype of the CD56+ cells was similar, showing an increase in the percentage of CD56+CD3- cells expressing CD69. CD69 is a differentiation antigen expressed shortly after the activation of T cells and NK cells, and is also thought to play a role in triggering NK cell cytotoxicity (374). Thus, increased expression of CD69 would be expected after activation of NK cells by interferon, as has been shown elsewhere (374). These results were strongly suggestive that interferon-a was the factor responsible for the activation of NK cells by CMV in this system. The induction of interferon in response to viral infection is considered to play an important part in the host’s antiviral immunity (439). The production of interferon by lymphocytes exposed to virus-infected target cells and the subsequent stimulation of NK cells by this interferon, has been proposed as the mechanism whereby NK cells preferentially lyse virus-infected rather than uninfected target cells (440). However, such a role for interferon has been challenged by some authors, who failed to show a correlation between the magnitude of target cell lysis by NK cells, and the levels of interferon detected in supernatant fluids after the incubation of HSV (441,442) and CMV (434) with PBMCs. Furthermore, the addition of neutralising antibodies specific for interferon to cytotoxicity assays, could not prevent the lysis of HSV-infected cells by NK cells (441,442). In the light of the results in the present study, the lack of correlation between the levels of interferon induced and target cell lysis, is unlikely to be relevant. Maximal NK cell activation

was observed here using concentrations lower than those compared in the study that failed to find a correlation between activation and interferon-a levels (434). This suggested that even the lower levels of interferon measured in the latter study had maximally activated NK cells, and therefore higher levels of interferon did not result in further activation. The pre-incubation of effector cells with interferon-a for 1 hr, before their use in 20 hr cytotoxicity assays, has been shown by others to increase the NK cell-mediated lysis of both uninfected and CMV-infected fibroblast target cells (415). In another study, the pre-treatment of PBM effector cells with 1000 lU/ml of interferon-a for 18 hr increased the NK cell-mediated lysis of uninfected and CMV-infected target cells, and reduced the time needed to measure the lysis of target cells in a cytotoxicity assay from 20 hr to 6 hr (431).

The results in this chapter show that the activation of NK cells by CMV is an indirect process. Virus particles, which need not necessarily be infectious, interact with the surface of, or are taken up by, DR+ accessory cells. In response to this interaction, these cells then produce interferon-a. The identity of the DR+, interferon-a producing cells was not investigated further in this study. However, recent reports have identified such cells, which produce interferon-a in response to a number of enveloped viruses, including HIV, HSV and vesicular stomatitis virus (443). These cells were a subset of immature DR+ dendritic cells, which expressed the receptor for IL-3 (GDI23) and CD4. They differed from mature dendritic cells by lacking the expression of the co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86) (444,445). These interferon-a producing cells were present at very low frequency (0.3%) in the peripheral blood, but were capable of producing large amounts of interferon-a after 6h of stimulation with ultraviolet-irradiated HSV-1 (444). It seems likely therefore, that the interferon-a production by DR+ cells in response to cell-free CMV that was observed here, was produced by the same interferon-a producing cells as those described by others.

The precise nature of this interaction between virus particles and interferon-a producing cells is unclear. However, there is evidence to suggest that interactions between viral proteins such as gp120 of HIV (446), glycoprotein D of HSV-1 (447) and glycoprotein M of coronavirus (448), and dendritic cells, stimulate the latter to

produce interferon-a. It is possible that these viruses induce interferon-a by simply