We report here that RSV infection of primary human BECs strongly up-regulated PD-L1 expression and that in CD8+ T cell/BEC co-cultures, RSV-infected BECs increased CD8+ T cell activation, proliferation, and antiviral cytokine and cytotoxic protein production. We observed the latter chiefly among effector-memory and terminally differentiated CD8+ T cell subsets. PD-L1 blockade enhanced CD8+ T cell secretion of IFN-γ, IL-2, and granzyme B and suppressed RSV replication. These data indicate that virus induction of PD-L1 expression on BECs can inhibit local CD8+ T cell antiviral activities in the context of an acute respiratory viral infection.
PD-L1 is constitutively expressed in primary nasal cells and BECs [18
]. Poly I:C and rhinovirus-16 induce both PD-L1 and PD-L2 on primary BECs and nasal epithelial cells [25
]. We found that RSV infection of primary BECs increased PD-L1-expression robustly, ~30-fold for mRNA and ~4-fold for protein. In contrast, rhinovirus-16 up-regulated PD-L1 protein only ~2-fold on human primary BECs [25
]. These data, together with the known inhibitory effects of PD-L1 in the context of chronic viral infections [2
], prompted us to investigate in vitro the functional importance of PD-L1 induction in the antiviral immune responses to RSV infection.
We first hypothesized that uninfected epithelial cells would not activate CD8+ T cells, but in contrast RSV-infected BECs would stimulate CD8+ T cells to express activation markers, proliferate, and induce cytokine and cytotoxic protein secretion.
CD69 is one of the earliest activation markers on T cells. The peak proportion of CD69+
T cells in response to alloantigen stimulation is reported to be <3% [26
]. Consistent with this, in our studies, CD69 was present on 2–3% of CD8+
T cells co-cultured with uninfected BECs. But this percentage was significantly increased (~3 times) on CD8+
T cells co-cultured with RSV-infected BECs. We noticed a weaker induction for CD25 and PD-1 (which is persistently up-regulated on exhausted lymphocytes in chronic viral infections) [2
]. PD-1 was found in low percentages (< 0.5%) on our CD8+
T cells as they were isolated from normal donors, and was not significantly increased on total CD8+
T cells co-cultured with RSV-infected BECs.
It is reported that healthy epithelium can suppress local lymphocyte activation, whereas infected epithelium becomes an activator [27
]. The mechanisms through which CD8+
T cells get activated by virus-infected BECs are not fully understood. It was shown that activation is partly contact-dependent and partly mediated by transforming growth factor β [27
]. We have observed that RSV up-regulates MHC class I molecule levels on the surface of infected BECs (data not shown). It is also likely that RSV up-regulates CD80 and CD86 molecules. These molecules have been shown to be induced by rhinovirus infection on respiratory epithelial cells [17
]. So far, there are no data to support that CD8+
T cell activation by virus-infected BECs occurs via these molecules.
T cells most likely found in contact with BECs in vivo are the EM and TD subpopulations [14
]. We observed that the levels of expression of CD69, CD25, and PD-1 were all higher in these subpopulations than in total CD8+
T cells co-cultured with uninfected BECs. In addition their induction by RSV-infected BECs was greater in the EM and TD subpopulations than in total CD8+
T cells. These data suggest the CD8+
T cells, which respond to RSV-infected BECs by activation, are the EM and TD CD8+
T cell subsets.
T cells did not proliferate in uninfected BEC/CD8+
T cell co-cultures. However, the majority of nonspecific-stimulated CD8+
T cells co-cultured with RSV-infected BECs underwent >1 round of proliferation. Nonspecific-stimulated CD8+
T cells cultured alone proliferated in a manner similar to CD8+
T cells co-cultured with RSV-infected BECs (data not shown). This proliferation suggests that un-infected epithelium actually has the capacity to inhibit CD8+
T cell proliferation, similar to results obtained in vitro with murine cells [27
Cytokine (IFN-γ and IL-2) and cytotoxic protein (granzyme and perforin) production showed similar patterns for the activation markers, with higher induction occurring in CD8+ T cells co-cultured with RSV-infected BECs compared with un-infected BECs. Induction of IFN-γ in CD8+ T cells occurred chiefly among the effector cell populations, whereas induction of IL-2 occurred mostly among naïve cells. Granzyme B and perforin production was found almost exclusively in EM and TD subpopulations. But CD8+ T cells expressed high levels of cytotoxic proteins, especially granzyme B, even when co-cultured with un-infected BECs. Thus, the increase in their numbers was relatively modest.
These data are in accordance with anticipated function: Resting BECs did not activate CD8+ T cells, but RSV-infected BECs equipped local CD8+ T cells for appropriate expansion and antiviral effector function.
We next hypothesized that increased PD-L1 expression in this context might prevent full activation of antiviral effector functions. Indeed, we found a further increase of IFN-γ, IL-2, and granzyme B levels in the presence of PD-L1 (but not PD-L2) blocking antibody, confirming that increased expression of PD-L1 acts as an inhibitor in this system.
Relatively few models have investigated functional memory T cell responses to live virus infections. We therefore developed this in vitro co-culture system to study these events in a system modeling epithelial/CD8+
T cell interactions as they might occur in vivo. T cells are found in association with epithelium in the respiratory tract, and it has been demonstrated that human bronchial epithelial cells can present antigens and directly activate cytotoxic CD8+
T cells [28
]. Substantial evidence underscores the fact that effector-memory CD8+
T cells do not proliferate in the lung or airways, suggesting that new cells continually come from the circulation [16
]. By using CD8+
T cells isolated from peripheral blood, we tried to create an in vitro environment similar to that found in the human respiratory tract. Our data confirm that PD-L1 can indeed inhibit local antiviral interactions between BECs and CD8+
T cells in acute respiratory infections.
These findings may have important implications in increased susceptibility to RSV infection in infants, as well as in incomplete memory immune responses to RSV in older children, adults, and the elderly. Blocking PD-L1 may be a useful therapeutic approach to augment antiviral immunity in acute respiratory viral infections such as RSV.