NFAT, bZIP proteins, and transcriptional programs in lymphocytes - ABSTRACT
Blocking antibodies to CTLA4, PD-1 and other inhibitory surface receptors expressed on exhausted T cells, or
blocking antibodies to the PD-1 ligands PD-L1 and PD-L2 expressed by tumor and stromal cells, have been
remarkably successful at promoting long-term tumor regression; moreover combinations of blocking antibodies
to multiple inhibitory receptors, often reinforced with activating antibodies to costimulatory receptors, have
been more effective than treatment with individual blocking antibodies alone. Despite these successes, many
patients still fail to respond to “immune checkpoint blockade” therapies, emphasizing the need to understand
immune cell “exhaustion” at a molecular level, both in mouse models and in humans.
The calcium- and calcineurin-regulated transcription factor NFAT is a driver of the transcriptional responses
underlying T cell activation. The T cell activation program mainly depends on cooperative binding of NFAT and
its transcriptional partner AP-1 (Fos-Jun) at composite DNA sites in gene promoters and enhancers. In parallel,
NFAT can activate a second transcriptional program that imposes a hyporesponsive state, typically termed
`exhaustion' or `dysfunction', that is observed in CD8+ T cells exposed to persistent antigen stimulation during
chronic viral infections and cancer. This second program is characterized by a spectrum of functionally
compromised states, including decreased cytokine expression and increased expression of multiple inhibitory
receptors (PD-1, CTLA4, LAG3, TIM3, TIGIT). Thus an effective alternative to combination therapies might be
to modulate the balance between NFAT-mediated programs of activation and exhaustion, so as to skew tumor-
infiltrating T cells away from exhaustion and towards effector function. We will test this hypothesis here.
Our experiments with an engineered NFAT1, minimally modified to prevent its interaction with AP1, have
established that the transcriptional program of exhaustion is independent of the NFAT1-AP1 interaction. We
have identified important targets of NFAT in the exhaustion program, including transcription factors of the Nr4a
family. Moreover, we have shown that Nr4a transcription factors act in part by repressing the expression or
activation of bZIP transcription factors that would otherwise promote an effector-like phenotype in exhausted
tumor-infiltrating T cells. In Aim 1, we will identify and characterize these bZIP transcription factors; in Aim 2,
we will define the differential roles of two NFAT family members, NFAT1 and NFAT2; in Aim 3, we will use
novel proteomic strategies to identify NFAT-interacting proteins that help impose the exhaustion program; and
in Aim 4, we will continue our successful search for small molecules that modulate the NFAT:AP-1 interaction.
Our proposed studies will test the hypothesis that CD8+ TILs are functionally silenced by a cell-intrinsic
transcriptional program mediated by NFAT activation and secondary repression of bZIP transcription factors.
The results will contribute to a broad mechanistic understanding of the transcriptional mechanisms operating in
mouse and human tumor-infiltrating T cells.
