Although the transcription factor retinoic acid receptor–related orphan receptor α (RORα) is required for development of innate lymphoid 2 cells (ILC2), it is highly expressed in other ILC subsets where its function remains unknown. In this Top Read, Lo et al. (p. 3209) used single-cell RNA analysis to define the role of RORα in ILC responses during chronic enteric infection. Analysis of RORα−deficient mice (Rorasg/sg) chronically infected with Salmonella revealed a reduction in the frequency of ILC3s by more than 3-fold in the cecum and the mesenteric lymph nodes (MLN). Furthermore, ILC3s present in the inflamed tissues of these mice showed decreased expression of key signature genes, including Rorc and activating cytokine receptors. Flow cytometric analysis supported these observations and showed a reduction in the frequency of RORγt+ ILC3s in the Salmonella-infected cecum and MLN of Rorasg/sg mice. Collectively, these data demonstrate that RORα plays an essential role in preserving functional ILC3s by modulating their ability to integrate environmental cues to efficiently produce cytokines.

In this Top Read, Zacharias and Legge (p. 3313) demonstrate that chronic ethanol (EtOH) consumption can impair CD8+ T cell memory in the lung. Mice were primed with influenza A virus (IAV), followed by 8 wk of either EtOH or water consumption, then challenged with IAV. Consumption of EtOH resulted in an 80% mortality rate following high dose IAV challenge, whereas mice receiving water survived secondary infection. The authors also found that EtOH consumption decreased both the lytic potential and the total number of Ag-experienced CD8+ T cells in the lung and draining lymph node. This decrease was consistent within the T resident memory (TRM), T central memory (TCM), and T peripheral memory (TPM) populations in the lung. TCM, TPM, and T effector memory (TEM) cells increased expansion within the lung vasculature of the EtOH treated mice in response to IAV–Ag challenge; however, within the lung tissue, TCM, TPM, and TEM showed little or no proliferation in response to IAV Ag. The authors also found that there is a reduction of CXCL10 and CXCL11in the lung tissue of EtOH–treated mice following IVA re-exposure, which is likely to impact the ability of memory CD8 T+ cells to migrate into lung tissue. Together, these data show that chronic EtOH consumption can disrupt established memory by reducing both the number and quality of CD8+ memory T cells, as well as decreasing their ability to migrate into the infected tissues.

Unlike the highly polymorphic MHC proteins, monomorphic molecules such as MR1, CD1d, and CD1b present Ags to T cells with species-wide TCR motifs. Previous CD1b tetramer studies identified a TCR motif in humans defined by the TCR β-chain variable gene 4-1 (TRBV4-1), which showed a low affinity binding to its antigenic target, as well as a less stringent TCR sequence conservation. In this Top Read, Reinink et al. (p. 3395) used CD1b tetramers with defined, but chemically diverse lipid Ags to demonstrate a connection between CD1b recognition and TRBV4-1 expression, thereby demonstrating a broad relationship between a TCR V region and its protein target. Crystal structures of two CD1b-specific TRBV4-1+ TCRs showed that germline-encoded residues in the CDR1 and CDR3 regions of TRBV4-1 represent sequences that interact with each other and consolidate the surface of the TCR. Mutational studies identified key positively charged residues within the TRBV4-1 TCR and key negatively charged residues in CD1b that are shared with CD1c, also recognized by TRBV4-1 TCRs. These data demonstrate that one TCR V region can mediate recognition of two related monomorphic Ag-presenting molecules independent of a defined lipid Ag.

In mice, the ability of naive T (TN) cells to respond to exogenous Ags correlates with the level of cross-reactivity against self-derived Ags, which can be quantified via the surrogate marker CD5. Accordingly, functional heterogeneity in murine CD8+ TN has also been identified by levels of CD5. In this Top Read, De Simone et al. (p. 3179) sought to determine if heterogeneity exists in human CD8+ TN cells. The authors identified two discrete populations of human CD8+ TN cells defined by the presence or absence of the chemokine receptor CXCR3. CXCR3+ TN cells displayed an effector-like transcriptional profile and expressed TCRs with physiochemical characteristics indicative of enhanced interactions with peptide–HLA class I Ags. These cells also frequently produced IL-2 and TNF in response to nonspecific activation and differentiated into Ag-specific effector cells in vitro. Further analysis of CXCR3+ TN cells showed that they were transcriptionally equivalent to murine CXCR3+ TN cells, which expressed high levels of CD5. Therefore, these findings demonstrate that humans harbor two distinct populations of CD8+ TN cells, supporting the hypothesis that T cell effector differentiation is shaped by heterogeneity in the preimmune repertoire of human CD8+ T cells.

In this Top Read, Zhao et al. (p. 3157) show that CCL2 is removed from the blood in a CCR2-dependent, but G-protein–independent manner. CCR2 antagonists are potential therapeutics for various chronic inflammatory disorders and cancer. Preclinical and clinical studies of CCR2 agonists have all demonstrated elevations in blood levels of CCL2, which complicates interpretation of in vivo responses to these potential therapeutics. Wild-type mice treated with CCR antagonists showed enhanced levels of circulating CCL2 that were similar to those seen in CCR2 knockout mice. In freshly isolated human peripheral blood monocytes incubated with CCR2 antagonists, accumulation of CCL2 positively correlated with antagonist dose and temperature. However, inhibition of G-protein signaling had no effect on accumulation of CCL2. Human embryonic kidney cells transfected with human CCR2 were able to clear CCL2 from the supernatant, but treatment with a CCR2 antagonist reversed this effect. The authors also demonstrated constitutive internalization of CCR2 that was not affected by antagonist treatment. Therefore, CCR2 antagonists block the interaction of CCL2 with CCR2, preventing uptake and thereby escalating CCL2 levels. This previously unknown mechanism may broadly impact interpretation of data from clinical studies using CCR2 antagonists.

In this Top Read, Anderson et al. (p. 3166) developed a method to edit autoimmune-associated genes in primary human T cells to accurately discern the impact of a candidate genetic variant on their function. Because experiments on bulk primary human T cells require near complete gene disruption, the authors used CRISPR/Cas9 to introduce a stop codon within candidate genetic loci. Using this method, Anderson et al. were able to demonstrate a >90% loss of ZAP70 and subsequent loss of response to TCR engagement. Ablation of either protein tyrosine phosphatase nonreceptor 22 (PTPN22) or protein tyrosine phosphatase nonreceptor 2 (PTPN2) has been shown to be a risk factor in various autoimmune diseases, although mouse and human data were sometimes conflicting. Disruption of PTPN22 in primary human CD4+ T cells led to hyperactive TCR signaling, while promoting IL-2 responsiveness and increased expression of PD-1, CD69, CD25, CD71, and suppressor of cytokine signaling 3 (SOCS3). A correlation between PTPN2 and SOCS3 was also seen in human subjects with the PTPN2 rs 1893217 risk allele. Together, these data indicate that optimized CRISPR/Cas9 gene editing is a robust platform to create homogenous, bulk primary human T cells for studying pathways that impact the loss of tolerance and identify potential therapeutic interventions.