In this study, we sought to determine the roles of the inhibitory receptors, PD-1 and CTLA-4, on T cell migration during the maintenance of tolerance. The results indicate that the disruption of PD-1-PD-L1 but not CTLA-4-B7 interactions enhances tolerized T cell interactions with antigen-bearing DCs, and facilitates the phosphorylation of key TCR signaling molecules. This T cell engagement with antigen-bearing DCs ultimately results in the production of effector cytokines and rapid progression of autoimmunity.
The duration of T cell-DCs contacts and its influence on T cell activation and the induction of tolerance has been a topic of great interest. Several other reports illustrated that during the induction of T cell tolerance both transient and stable DC interactions can occur 20,23
. During the process of T cell activation the duration of T cell-DC contacts is highly variable in vivo
, ranging from minutes to several hours 39
. Early reports suggested that initial interactions during the first phase of T cell activation tend to be transient [5-10 minutes during the first 3-15 hours 20,21,40
]. However, a more recent report found that long lived T cell-DC interactions could occur following the initial T cell-DC contact and that prolonged interactions were required for T cell activation 41
. In this study, Erk phosphorylation occurred early after stable T cell-DC conjugates were formed, and IFN-γ production increased with longer T cell-DC interactions 41
. These interactions required TCR engagement of peptide-MHCII complexes, as MHCII blockade in vivo
terminated stable contacts, increased T cell motility, decreased T cell proliferation and prevented IFN-γ production. The second phase subsequently results in longer lived contacts where T cells form stable conjugates and begin to secrete cytokines which transitions into a third phase of high motility and rapid proliferation 21
. Our studies demonstrated that PD-1 blockade restored stable T cell-DC contacts, Erk phosphorylation, IFN-γ production and, most importantly, T1D. These findings suggest that PD-1 normally functions to prevent the T cell stop signal and the formation of stable conjugates with antigen bearing DCs.
Anergic T cells have been shown in vitro
to form unstable immunological synapses with allogeneic APCs and failed to recruit the signaling proteins necessary to initiate T-cell activation 42
. We suggest that these transient interactions are required in our system (but impossible to discern in vivo
), since the breakdown of tolerance following PD-1-PD-L1 blockade only occurs when antigen is present, as in the case of PLN and pancreas, and does not occur in the ILN. Thus, abrogation of tolerance allows the stabilization of these interactions allowing full T cell activation and clinical disease.
Recent reports suggested that B7-1 and PD-L1 interact directly with each other to negatively regulate T cells 13
. Thus far, however, in vivo
data to support the functionality of this interaction have not been reported. Our data do not suggest that this interaction was responsible for the tolerant phenotype observed in this study, as administration of anti-B7-1 had no effect on T cell velocity, track displacement, or movement trajectories when compared to isotype control antibody (data not shown). In addition, PD-1 blocking antibodies induced effects similar to those induced by PD-L1 blockade (data not shown). Finally, tolerized BDC2.5 T cells arrested and stopped when transferred to the PD-L1 deficient (PD-L1-/-
) recipients similar to that observed in experiments presented here using PD-L1 antibody blockade, supporting the critical role for PD-1-PD-L1 interactions for tolerance (data not shown).
Blockade of CTLA-4–B7 interactions can prevent induction of tolerance by peptide-pulsed fixed APCs but could not reverse tolerance once established 10
. It is important to note that the same dose of blocking CTLA-4–specific antibody was used in these MPLSM experiments as in a previous study that documented a critical role for CTLA-4 during the induction of tolerance 10
. We detected no influence of CTLA-4 in the migratory behavior of tolerized T cells. Due to the complex nature of lattice formation with B7-1, we have also explored anti-CTLA-4 Fab fragments with similar results as shown using intact anti-CTLA-4. The results presented here suggest that CTLA-4 inhibition had different qualitative and quantitative biological consequences than PD-1-PD-L1 blockade.
Although CTLA-4 and PD-1 both limit T cell signaling, cytokine production, cell cycle progression, and may share potential targets, some key biochemical differences have been reported. Upon PD-L engagement, PD-1 can bind SH2-domain containing tyrosine phosphatase 1 (SHP-1) and SHP-2 4,43
. The binding of SHP-1 and SHP-2 can terminate early TCR signals by dephosphorylating key signaling intermediates including the kinases Akt, PI3K, ZAP-70, and PKC-θ. Like PD-1, CTLA-4 can interact with SHP-1 and SHP-2 14
. Unlike PD-1, CTLA-4 can also interact with the phosphatase PP2a 15
. Another difference between these two inhibitory receptors is the structural motif used to bind phosphatases. CTLA-4 interacts through the immunoreceptor tyrosine-based inhibitory motifs (ITIM) while PD-1 contains an additional motif, the immunoreceptor tyrosine-based switch motif (ITSM) 43
. Mutation of the ITIM motif had little effect on signaling, while ITSM mutations abrogated the ability of PD-1 to limit T cell population expansion 43
. This suggests that PD-1 and CTLA-4 use different structural motifs to bind and recruit phosphatases for signal blockade. Future work is necessary to determine the precise biochemical relationship between these two potent negative regulatory molecules.
Genetic experiments may help to explain the different roles for PD-1 and CTLA-4 in immune homeostasis, breakdown of tolerance and establishment of autoimmunity. CTLA-4 deficiency results in rapid multi-organ tissue inflammation and death within 3-4 weeks of age, regardless of mouse genetic background 5
, whereas autoimmunity in PD-1-deficient mice is slower and tissue specific in a manner dependent on genetic background 4
. These reports suggest that deficiencies in PD-1/PD-L1 pathway may potentiate tissue specific autoimmune predispositions. CTLA-4 on the other hand, controls multi-organ infiltrate and autoimmunity irrespective of genetic background. CTLA-4 and PD-1 ligand expression and distribution may help explain these differences. The fundamental difference between the effects of CTLA-4 and PD-1 on T cell migration as described here may also help to explain these differences.
Two recent reports investigated the role of CTLA-4 on T cell stop signals 44,45
. One study found that CTLA-4 positive T cells failed to stop or slow down in response to in vivo
peptide challenge, and anti-CTLA-4 increased T cell motility 44
. A follow-up study reported that CTLA-4-deficient T cells showed a marked resistance to a stop-signal induced by anti-CD3 45
. It is difficult to explain the discrepancies between these previous studies, but it may be due to subtle differences between sorted T cell subpopulations, CTLA-4 surface stability, blocking antibodies, or use of knockout T cells. Here we found no influence of CTLA-4 blockade on TCR-driven stop signals. In our study, CTLA-4 blockade did not alter the DC-binding properties of the tolerant T cells, did not result in significant ERK phosphorylation, did not restore IFN-γ production, and did not result in the rapid development of autoimmune diabetes. One major difference is the previous reports tracked the migration of naïve T cells during primary stimulation 44,45
whereas the present studies focused on anergic T cells during the re-activation phase.
Finally, it is interesting to note that PD-1-PD-L1 blockade resulted in increased accumulation and/or enhanced proliferation of antigen-specific T cells within the target tissue 10
. This finding supports a key role for T cell-DC interactions during tissue-specific reactivation. Future work in this area will determine if CD4+
T cells interact directly with islet target cells or through a tissue-specific MHC II+
DC. Further investigation of the signals that maintain tolerance in this and similar settings will aid in our understanding of how to exploit the PD-1-PD-L1 pathway in efforts to prevent and treat autoimmunity, promote transplant acceptance, and limit tumor growth.