A central tenet of the clonal selection theory (CST), the current cornerstone of adaptive immunity, states that naïve T cells are selected on the basis of specificity to antigen and that the rest of the repertoire remains agnostic to antigen challenge. However, CST and more recent paradigms have not considered the need for negative regulatory signals to maintain quiescence of the unselected T cells as a pivotal aspect of maintaining clonal selection. Silencing of naïve T cells, or tolerance, also becomes critical upon encounter with self-antigen, whereby self-reactive clones cannot be allowed to develop effector function. These events are known to be interrelated because the loss of quiescence precipitates a loss in tolerance.

V-type immunoglobulin domain-containing suppressor of T cell activation (VISTA) is an inhibitory receptor expressed on naïve T lymphocytes, and genetic deletion of VISTA culminates in T cell–mediated autoimmunity. Unlike other negative checkpoint regulators identified to date, VISTA is expressed on naïve T cells, which led us to investigate its function in maintaining naïve T cell quiescence and tolerance.

Using a combination of single-cell RNA sequencing and single-cell ATAC sequencing technologies coupled with in vivo analyses, we investigated the cell-state phenotype of naïve CD4⁺ (CD44^lo CD62L^hi) T cells in mice and found multiple T cell subpopulations exist within a surprisingly heterogeneous naïve T cell compartment. The vast majority of cells display a quiescent phenotype. However, there were subsets expressing less quiescent, memory-like features, a cluster with a high interferon-I (IFN-I) response signature, and a population with higher early T cell receptor (TCR) signaling genes. We show that genetic deletion of VISTA results in a major redistribution of the naïve T cell subsets with the notable reduction of the quiescent subset and the enhanced presence of a memory-like activated T cell subset. In the absence of intrinsic VISTA expression, naïve T cells were more epigenetically and transcriptionally primed toward stronger responses to TCR and cytokine stimulation, providing a rationale for the enhancement of CD44^hi CD4⁺ memory-like T cells observed in the absence of VISTA. As a consequence of reduced quiescence within the naïve T cell compartment, these naïve T cells were less susceptible to tolerance induction. Genetic deletion or antibody blockade of VISTA resulted in significant expansion and reduced tolerance of antigen-specific T cells. These effects were abolished under inflammatory conditions where VISTA expression on antigen-specific T cells was reduced. By contrast, agonistic engagement of VISTA and antigen exposure increased tolerance induction by enhancing the death and deletion of antigen-specific T cells. Tolerogenic death induced by anti-VISTA was demonstrated in a model of acute graft-versus-host disease (GVHD), whereby deletion of donor host reactive T cells resulted in a pronounced therapeutic benefit and long-term survival. The gene signature of VISTA^−/− T cells overlapped significantly with T cells from patients with the autoimmune diseases systemic lupus erythematosus and rheumatoid arthritis, suggesting that VISTA may play a broad regulatory role in suppressing T cell self-reactivity.

We introduce VISTA as the earliest checkpoint regulator of peripheral T cell tolerance identified to date and describe VISTA as the first of a new class of immunoregulatory molecules whose function is to enforce quiescence in naïve T lymphocytes. In addition, comprehensive epigenetic and transcriptional analyses defined fluorescence …