The factors that lead to the development of PML in individuals treated with natalizumab need to be investigated in more detail, particularly immune responses to the virus. Consequently, we investigated the T cell immune response to the entire JC virus proteome longitudinally in subjects with MS who were initiating therapy with natalizumab and in subjects who had natalizumab-associated PML. The principal findings were: 1) T cell responses were identified against all JC virus proteins and could be measured ex vivo in the peripheral blood of individuals treated with natalizumab and healthy subjects; 2) the magnitude and quality of T cell responses to JCV and CMV did not change from baseline through the first several months of natalizumab treatment; 3) the frequency of T cells producing the cytokine IL-10 in response to mitogenic stimulation temporarily increased after the first dose of natalizumab; 4) individuals with PML either made no detectable T cell responses to JCV or had JCV-specific CD4 T cell responses uniquely dominated by IL-10 production rather than IFNγ; and 5) individuals shortly after PML diagnosis had higher levels of IL-10, IL-5 and IL-15 in the CSF.
Whether JCV-specific T cell responses can be reliably measured ex vivo
and the effect of natalizumab on these responses 
have both been the subject of some debate. Using ex vivo
stimulation with overlapping peptides, we readily detected CD4 and CD8 T cell responses to JCV by multiparameter ICS in healthy subjects, subjects with MS and in some subjects with PML. This approach could be useful in monitoring JCV-specific T cell function in natalizumab-treated individuals in the context of a vaccine or a risk stratification protocol. Our findings that JCV-specific T cells are directed against each of the viral proteins and that the specificity and immunodominance of the response varies among individuals strongly suggest that measuring responses to all viral proteins rather than VP1 alone is essential to obtaining a complete picture of JCV-specific immunity. Our results also suggest that measuring multiple cytokines rather than IFNγ alone allows for the identification of associations that would otherwise be missed, and in particular highlight the potential importance of IL-10 in evaluating T cell responses to JCV. In our longitudinal study we found no difference in either the magnitude or functional profile of the total JCV-specific T cell response during short-term natalizumab treatment. Although the sample size of our study was limited in power to detect longitudinal differences, the fact that no difference was observed in any of the cytokines measured, or in their relative contribution to the response at different time points, suggests that JCV-specific T cell responses are not altered simply by the initiation of natalizumab.
However, in the subjects with PML, JCV-specific CD4 T cell responses were either undetectable or uniquely dominated by IL-10. Importantly, it has been shown that IL-10 is detrimental to the clearance of lymphocytic choriomeningitis virus (LCMV) infection because of its inhibitory effect on virus-specific memory CD4 T cells 
. Furthermore, vaccines which stimulate CD4 T cell IL-10 production can limit the elicitation of protective polyfunctional CD4 T cells 
. Thus, the production of IL-10 but not IFNγ, TNF or IL-2 by JCV-specific CD4 T cells may interfere with antiviral activity to the detriment of control of JCV replication, either locally in the CNS or in peripheral tissues, and may consequently be causative of PML. Another interpretation is that IL-10 production, which was not detected until >4 months after the subjects had developed PML, is a later-stage response to the inflammation often associated with PML (PML-IRIS). This sample size does not allow us definitively to link the time since diagnosis to the presence of an IL-10 response. However, our finding that IL-10 was detectable in 50% of early CSF samples suggests that IL-10 production may occur early in natalizumab-associated PML disease. Importantly, these two interpretations are not mutually exclusive. Although the role of IL-10 in PML associated with other conditions has not been thoroughly characterized, one study found increased JCV-specific IL-10 production by total PBMC from HIV+
PML cases, but not from non-HIV PML cases 
. This result suggests that IL-10 may be associated with PML resulting from causes other than natalizumab therapy.
Notably, we found that the frequency of memory CD4 T cells that produce IL-10 upon mitogenic stimulation is transiently increased after the first dose of natalizumab. It is tempting, therefore, to speculate that natalizumab may skew the CD4 T cell response toward IL-10 production and away from production of IFNγ, TNF, and IL-2. This suggests a possible mechanism by which natalizumab treatment could lead to PML, as 50% of the subjects with natalizumab-associated PML that we studied produced IL-10 in response to JCV, and the other 50% had no measurable T cell response to the virus. There are a number of mechanisms by which natalizumab treatment could potentially skew the CD4 T cell response toward IL-10 production, including increasing antigen load through mobilization of infected CD34+
, which may affect the cytokine profile 
, altering the antigen-presenting cells (APCs) that interact with the T cells 
, or altering T cell trafficking and thus with which APCs the T cells interact 
. There is also the possibility that a direct effect of natalizumab on T cells could affect cytokine production, as may occur with ribavirin treatment for hepatitis C virus 
The low probability that any of the natalizumab-treated MS study subjects could go on to develop PML is consistent with the lack of JCV-specific IL-10 production in those subjects who did not have PML. Furthermore, in this study we were only able to measure T cell responses in the peripheral blood and were not able to sample the CNS of subjects without PML. A previous study showed that after 12 months of natalizumab treatment, levels of IL-10 mRNA were increased in bulk CSF cells, while remaining unchanged in PBMC 
. Although this finding may be due to altered CSF cell subset composition after treatment rather than upregulation of IL-10, it supports the hypothesis that natalizumab may alter the cytokine milieu in the CNS. Indeed, we found increased levels of IL-10 and IL-5 in CSF samples from individuals with natalizumab-associated PML. Although these cytokines are not typically associated with control of virus replication or with each other, their aberrant production may be indicative of an immune response that fails to control JCV replication at the site of disease. The increased CSF IL-15 we observed early in PML disease is consistent with inflammatory CNS disease 
but is unlikely to be due solely to MS disease-associated inflammation 
as the PML and non-PML groups both consisted of individuals with MS who were treated with natalizumab and had neurological symptoms consistent with PML. Thus far, it is not possible to demonstrate a causative link between IL-10 production and PML as the very low incidence of natalizumab-associated PML makes a prospective study unlikely. However, we believe that the potential mechanism suggested by these data should inform future work.
Taken together, our data provide a framework for understanding immune control of JC viremia and the development of PML and suggest avenues of investigation to allow the better monitoring, treatment and prevention of PML in natalizumab-treated people. First, our finding that subjects with PML lacked JCV-specific T cell responses or produced IL-10 in response to stimulation suggests that immune monitoring might identify natalizumab-treated individuals who are at risk of developing PML, by screening subjects prior to treatment or while on treatment. None of the MS patients without PML or healthy subjects included in our study showed an absent or IL-10 producing T cell response similar to that observed in the subjects with PML, and this suggests that individuals with these phenotypes are relatively rare and could be identified by immune monitoring prior to treatment. The potential of such screening of JCV-specific T cell responses to identify a small number of individuals at risk for the development of PML could be complementary to stratification strategies based on antibody levels that are currently being tested to identify approximately 50% of treated individuals who are at increased risk 
. Second, the unique IL-10 response to JCV in two PML cases and the increased levels of IL-10 in the CSF of subjects with PML suggests that IL-10 or the IL-10 receptor may be potential therapeutic targets in natalizumab-associated PML 
. Finally, the poor magnitude or quality of the memory T cell response to JCV in subjects with PML suggests that a vaccine which boosts JCV-specific T cells that produce IFNγ, TNF and IL-2 could play a role in the prevention of natalizumab-associated PML.