Supplementary Materials1. growth and pinpointed when lineage choice events occurred during differentiation. Comparable alterations in T cell phenotypes were observed following anti-CTLA-4 and anti-PD-1 antibody blockade. These findings implicate unfavorable costimulation as a key regulator and determinant of T cell differentiation, and suggest that checkpoint blockade may work in part by altering the limits of T cell phenotypes. Graphical Abstarct eTOC blurb Unfavorable costimulation is a critical regulator of T cell activity. Wei et al. characterize T cells arising in CTLA-4- and PD-1-deficient mice using mass cytometry and computational methods. They show that these unfavorable costimulatory molecules impose boundaries on T cell phenotypes during peripheral differentiation, suggesting that checkpoint blockade may work in part by altering the limits of T cell phenotypes. Introduction Unfavorable costimulation of T cells, mediated by molecules such as CTLA-4 and PD-1, maintains T cell activity within a desired physiological window, enabling effective acknowledgement of foreign antigens while restraining aberrant responses against self-antigens (Chen and Flies, 2013; Pardoll, 2012). In addition, peripheral D-AP5 differentiation generates a wide range of specialized T cell subsets that respond to diverse immunological difficulties (O’Shea and Paul, 2010; Zhou et al., 2009). How T cell differentiation is usually regulated by diverse cellular inputs remains unclear. T cell receptor transmission strength and cytokine signaling are recognized as key determinants of T cell differentiation (Zhou et al., 2009), but how other important signals regulate T cell differentiation remains unknown. In particular, the role of T cell costimulation in T cell differentiation remains unclear despite its well-established functional role in T cell activation. Thus, we sought to determine whether unfavorable costimulation has a functional role in both T cell activation as well as differentiation. CD28 is the primary source of positive costimulation and represents a critical second transmission for T cell activation following T cell receptor (TCR) engagement (Chen and Flies, 2013). Upon ligation by D-AP5 B7 ligands (B7-1 or B7-2), CD28 signals through phosphoinositide 3-kinase (PI3K) to reinforce downstream activation pathways. TCR engagement in the absence of CD28 costimulation leads to T cell anergy, a state of unresponsiveness. Ligation of CD28 prevents the induction of anergy in the absence of costimulation (Harding et al., 1992). Thus, effective priming of T cell activation requires cell extrinsic costimulation by B7 ligand expressing antigen presenting cells (APC). CTLA-4 principally acts to regulate T cell activation by competing D-AP5 with CD28 and thus, limiting positive costimulation (Chen and Flies, 2013; Pardoll, 2012). CTLA-4 expression is detected within 1 hour of T cell Mouse monoclonal to BNP activation, reaches peak levels within approximately 48 hours, and is trafficked to the immunological synapse to rapidly attenuate T cell activation (Egen and Allison, 2002; Lindsten et al., 1993; Walunas et al., 1994). Because CTLA-4 has higher affinity and avidity for B7 than CD28, CTLA-4 competitively inhibits CD28-mediated positive costimulation (Engelhardt et al., 2006; Pentcheva-Hoang et al., 2004; van der Merwe et al., 1997). It has also been reported that CTLA-4 can take action via removal of B7 ligands from APCs (Hou et al., 2015; Qureshi et al., 2011), regulation of T cell motility (Schneider et al., 2006), cell extrinsic suppression by T regulatory (Treg) cells (Wing et al., 2008), and cell intrinsic effects on signaling (Lee et al., 1998). Furthermore, mutant versions of CTLA-4, which ablate cytoplasmic tail domain name function, exhibit only partial activity (Carreno D-AP5 et al., 2000; Masteller et al., 2000). Together, these findings demonstrate that CTLA-4 regulates T cell activation via multiple unique mechanisms but also highlight our incomplete understanding of CTLA-4 biology. We sought to understand whether in addition to its role in attenuating activation, CTLA-4 also has a D-AP5 related but unique function in regulating T cell differentiation. As T.