Mechanistic understanding of the inhibitory immunoreceptor PD-1 is largely based on mouse models, but human and mouse PD-1 share only 59.6% amino acid identity. Here, we found that human PD-1 is more inhibitory than mouse PD-1, owing to stronger interactions with the ligands PD-L1 and PD-L2 and more efficient recruitment of the effector phosphatase Shp2. In a mouse melanoma model with adoptively transferred T cells, humanization of a PD-1 intracellular domain disrupted the antitumor activity of CD8+ T cells and increased the magnitude of anti-PD-1 response. We identified a motif highly conserved across vertebrate PD-1 orthologs, absent in rodents, as a key determinant for differential Shp2 recruitment. Evolutionary analysis suggested that PD-1 underwent a rodent lineage-specific functional attenuation during evolution. Together, our study uncovers species-specific features of the PD-1 pathway, with implications for PD-1 evolution and differential anti-PD-(L)1 responses in mouse models and human patients.

Mechanistic understanding of the immune checkpoint receptor PD1 is largely based on mouse models, but human and mouse PD1 orthologs exhibit only 59.6% identity in amino acid sequences. Here we show that human PD1 is more inhibitory than mouse PD1 due to stronger interactions with the ligands PDL1 and PDL2 and with the effector phosphatase Shp2. A novel motif highly conserved among PD1 orthologs in vertebrates except in rodents is primarily responsible for the differential Shp2 recruitment. Evolutionary analysis suggested that rodent PD1 orthologs uniquely underwent functional relaxation, particularly during the K-Pg boundary. Humanization of the PD1 intracellular domain disrupted the anti-tumor activity of mouse T cells while increasing the magnitude of anti-PD1 response. Together, our study uncovers species-specific features of the PD1 pathway, with implications to PD1 evolution and differential anti-PD(L)1 responses in mouse models and human patients.

Mucosal-associated invariant T (MAIT) cells are a subset of T lymphocytes that respond to microbial metabolites. We defined MAIT cell populations in different organs and characterized the developmental pathway of mouse and human MAIT cells in the thymus using single-cell RNA sequencing and phenotypic and metabolic analyses. We showed that the predominant mouse subset, which produced IL-17 (MAIT17), and the subset that produced IFN-γ (MAIT1) had not only greatly different transcriptomes but also different metabolic states. MAIT17 cells in different organs exhibited increased lipid uptake, lipid storage, and mitochondrial potential compared with MAIT1 cells. All these properties were similar in the thymus and likely acquired there. Human MAIT cells in lung and blood were more homogeneous but still differed between tissues. Human MAIT cells had increased fatty acid uptake and lipid storage in blood and lung, similar to human CD8 T resident memory cells, but unlike mouse MAIT17 cells, they lacked increased mitochondrial potential. Although mouse and human MAIT cell transcriptomes showed similarities for immature cells in the thymus, they diverged more strikingly in the periphery. Analysis of pet store mice demonstrated decreased lung MAIT17 cells in these so-called "dirty" mice, indicative of an environmental influence on MAIT cell subsets and function.

In response to an intracellular infectious agent, the immune system produces a specific cellular response as well as a T cell-dependent Ab response. Precursor T cells differentiate into effector T cells, including Th1 cells, and T follicular helper (TFH) cells. The latter cooperate with B cells to form germinal centers and induce the formation of Ab-forming plasmacytes. One major focal point for control of T cell differentiation is the transcription factor BCL6. In this study, we demonstrated that the Bcl6 gene is regulated by FOXO1-binding, cis-acting sequences located in a highly conserved region of the first Bcl6 intron. In both mouse and human T cells, deletion of the tandem FOXO1 binding sites increased the expression of BCL6 and enhanced the proportion of TFH cells. These results reveal a fundamental control point for cellular versus humoral immunity.

CRISPR-based active genetic elements, or gene-drives, copied via homology-directed repair (HDR) in the germline, are transmitted to progeny at super-Mendelian frequencies. Active genetic elements also can generate widespread somatic mutations, but the genetic basis for such phenotypes remains uncertain. It is generally assumed that such somatic mutations are generated by non-homologous end-joining (NHEJ), the predominant double stranded break repair pathway active in somatic cells. Here, we develop CopyCatcher systems in Drosophila to detect and quantify somatic gene conversion (SGC) events. CopyCatchers inserted into two independent genetic loci reveal unexpectedly high rates of SGC in the Drosophila eye and thoracic epidermis. Focused RNAi-based genetic screens identify several unanticipated loci altering SGC efficiency, one of which (c-MYC), when downregulated, promotes SGC mediated by both plasmid and homologous chromosome-templates in human HEK293T cells. Collectively, these studies suggest that CopyCatchers can serve as effective discovery platforms to inform potential gene therapy strategies.

Naive and memory T cells are maintained in a quiescent state, yet capable of rapid response and differentiation to antigen challenge via molecular mechanisms that are not fully understood. In naive cells, the deletion of Foxo1 following thymic development results in the increased expression of multiple AP-1 family members, rendering T cells less able to respond to antigenic challenge. Similarly, in the absence of FOXO1, post-infection memory T cells exhibit the characteristics of extended activation and senescence. Age-based analysis of human peripheral T cells reveals that levels of FOXO1 and its downstream target, TCF7, are inversely related to host age, whereas the opposite is found for AP-1 factors. These characteristics of aging also correlate with the formation of T cells manifesting features of cellular senescence. Our work illustrates a role for FOXO1 in the active maintenance of stem-like properties in T cells at the timescales of acute infection and organismal life span.

In response to infection, pathogen-specific CD8 T cells differentiate into functionally diverse effector and memory T cell populations critical for resolving disease and providing durable immunity. Through small-molecule inhibition, RNAi studies, and induced genetic deletion, we reveal an essential role for the chromatin modifier and BET family member BRD4 in supporting the differentiation and maintenance of terminally fated effector CD8 T cells during infection. BRD4 bound diverse regulatory regions critical to effector T cell differentiation and controlled transcriptional activity of terminal effector-specific super-enhancers in vivo. Consequentially, induced deletion of Brd4 or small molecule-mediated BET inhibition impaired maintenance of a terminal effector T cell phenotype. BRD4 was also required for terminal differentiation of CD8 T cells in the tumor microenvironment in murine models, which we show has implications for immunotherapies. Taken together, these data reveal an unappreciated requirement for BRD4 in coordinating activity of cis regulatory elements to control CD8 T cell fate and lineage stability.

Highly efficient gene conversion systems have the potential to facilitate the study of complex genetic traits using laboratory mice and, if implemented as a "gene drive," to limit loss of biodiversity and disease transmission caused by wild rodent populations. We previously showed that such a system of gene conversion from heterozygous to homozygous after a sequence targeted CRISPR/Cas9 double-strand DNA break (DSB) is feasible in the female mouse germline. In the male germline, however, all DSBs were instead repaired by end joining (EJ) mechanisms to form an "insertion/deletion" (indel) mutation. These observations suggested that timing Cas9 expression to coincide with meiosis I is critical to favor conditions when homologous chromosomes are aligned and interchromosomal homology-directed repair (HDR) mechanisms predominate. Here, using a Cas9 knock-in allele at the Spo11 locus, we show that meiotic expression of Cas9 does indeed mediate gene conversion in the male as well as in the female germline. However, the low frequency of both HDR and indel mutation in both male and female germlines suggests that Cas9 may be expressed from the Spo11 locus at levels too low for efficient DSB formation. We suggest that more robust Cas9 expression initiated during early meiosis I may improve the efficiency of gene conversion and further increase the rate of "super-mendelian" inheritance from both male and female mice.

T cells able to control neoplasia or chronic infections display a signature gene expression profile similar or identical to that of central memory T cells. These cells have qualities of self-renewal and a plasticity that allow them to repeatedly undergo activation (growth, proliferation, and differentiation), followed by quiescence. It is these qualities that define the ability of T cells to establish an equilibrium with chronic infectious agents, and also preserve the ability of T cells to be re-activated (by checkpoint therapy) in response to malignant cancers. Here we describe distinctions between the forms of inhibition mediated by tumors and persistent viruses, we review the properties of T cells associated with long-term immunity, and we identify the transcription factor, FOXO1, as the control point for a program of gene expression that allows CD8(+) T cells to undergo serial reactivation and self-renewal.

Memory CD8 T cells provide durable protection against diverse intracellular pathogens and can be broadly segregated into distinct circulating and tissue-resident populations. Paradigmatic studies have demonstrated that circulating memory cells can be further divided into effector memory (TEM) and central memory (TCM) populations based on discrete functional characteristics. Following resolution of infection, we identified a persisting antigen-specific CD8 T cell population that was terminally fated with potent effector function but maintained memory T cell qualities and conferred robust protection against reinfection. Notably, this terminally differentiated effector memory CD8 T cell population (terminal-TEM) was conflated within the conventional TEM population, prompting redefinition of the classical characteristics of TEM cells. Murine terminal-TEM were transcriptionally, functionally, and developmentally unique compared to TEM cells. Through mass cytometry and single-cell RNA sequencing (RNA-seq) analyses of human peripheral blood from healthy individuals, we also identified an analogous terminal-TEM population of CD8 T cells that was transcriptionally distinct from TEM and TCM. Key findings from this study show that parsing of terminal-TEM from conventionally defined TEM challenge the reported characteristics of TEM biology, including enhanced presence in lymphoid tissues, robust IL-2 production, and recall potential, greater than expected homeostatic fitness, refined transcription factor dependencies, and a distinct molecular phenotype. Classification of terminal-TEM and clarification of TEM biology hold broad implications for understanding the molecular regulation of memory cell states and harnessing immunological memory to improve immunotherapies.

Generation of T-cell memory is crucial in conferring vaccine-induced immunity, particularly against pathogens where neutralizing antibodies alone are insufficient at providing long-term protection. While great advances have been made in understanding the generation and maintenance of memory CD8+ T cells and B cells, mechanisms underlying the generation of memory CD4+ T cells have remained relatively elusive. This limitation is in part due to the multi-potency and lineage plasticity exhibited by CD4+ T helper (TH) cells. Using the LCMV viral infection model, we show that the T follicular helper (TFH) subset are the predominant CD4+ memory T cell type based on its relative abundance following pathogen elimination as well as its multi-potent potential upon antigen re-challenge. This multi-potency of TFH memory cells during secondary challenge has also been observed in influenza infection and acute bacterial infection, suggesting that the TFH memory subset is most capable of providing a comprehensive and robust secondary response. From advancements in CD8+ memory T cell studies, it has become clear that transcription factors (TF) serve as crucial arbiters for the cell-fate decisions between short-lived effector and memory. To investigate the transcriptional mechanisms underlying TFH formation, we employed a novel bioinformatics analysis utilizing the PageRank algorithm that combines both RNA-seq and ATAC-seq and have identified putative transcriptional regulators of memory TFH differentiation. Further understanding of TFH memory formation will undoubtedly unveil new insights into CD4+ T cell memory in hopes of improving vaccine-based immunity.

Memory CD8+ T cells afford long-lived, durable protection against infection and malignancy. Despite the critical relevance of memory T cells to vaccination and immunotherapies, the transcriptional and epigenetic signals instructing memory T cell fate remain unclear. Here, we utilized a pooled RNA interference screen to evaluate the activity of transcription factors and chromatin modifiers governing memory T cell differentiation in vivo. The epigenetic ‘reader,’ Brd4 of the BET protein family, emerged as a top regulator of memory T cell differentiation. Through RNA interference, small molecule inhibition and inducible genetic deletion, we established a central role for Brd4 in mediating CD8+ T cell proliferation, differentiation and function during acute viral infection. Brd4 was required for optimal expression of fate-specifying transcription factors, and a deficiency of Brd4 activity resulted in diminished formation of effector memory and CD103+ tissue-resident memory CD8+ T cells. Given that BET inhibition has emerged as a powerful approach for suppressing tumor growth, we also evaluated how BET inhibition and Brd4-deficiency influence T cell activity during cancer. Brd4 was required for intratumoral accumulation and function of T cells in a mouse model of melanoma, and BET inhibition suppressed T cell-mediated control of tumor growth. However, epigenetic targeting of Brd4 constrained terminal differentiation of T cells within tumors, biasing tumor-resident T cells towards a reprogrammable TCF1-expressing phenotype. These studies establish a novel role for Brd4 in CD8+ T cell biology and provide insight for immunotherapy approaches designed to leverage the dynamic activity of CD8+ T cells in tumors.

Immunity following an acutely resolved infection or the long-term equipoise of chronic viral infections often depends on the maintenance of antigen-specific CD8(+) T cells, yet the ongoing transcriptional requirements of these cells remain unclear. We show that active and continuous programming by FOXO1 is required for the functional maintenance of a memory population. Upon Foxo1 deletion following resolution of an infection, memory cells rapidly lost their characteristic gene expression, gradually declined in number, and were impaired in self-renewal. This was extended to chronic infections, as a loss of FOXO1 during a persistent viral infection led to a rapid decline of the TCF7 (a.k.a. TCF1)-expressing memory-like subset of CD8+ T cells. We further establish FOXO1 regulation as a characteristic of human memory CD8(+) T cells. Overall, we show that the molecular and functional longevity of a memory T cell population is actively maintained by the transcription factor FOXO1.

Caspase-8 (CASP8) is known as an executioner of apoptosis, but more recent studies have shown that it participates in the regulation of necroptosis and innate immunity. In this study, we show that CASP8 negatively regulates retinoic acid-inducible gene I (RIG-I) signaling such that, in its absence, stimulation of the RIG-I pathway in dendritic cells (DCs) produced modestly enhanced activation of IFN regulatory factor 3 with correspondingly greater amounts of proinflammatory cytokines. In addition, mice lacking DC-specific CASP8 (dcCasp8(-/-) mice) develop age-dependent symptoms of autoimmune disease characterized by hyperactive DCs and T cells, spleen and liver immunopathology, and the appearance of Th1-polarized CD4(+) T cells. Such mice infected with chronic lymphocytic choriomeningitis virus, an RNA virus detected by RIG-I, mounted an enhanced lymphocytic choriomeningitis virus-specific immune response as measured by increased proportions of Ag-specific CD4(+) T cells and multicytokineproducing CD4(+) and CD8(+) T cells. These results show that CASP8 subtly modulates DC maturation, which controls the spontaneous appearance of autoimmune T cells while simultaneously attenuating the acquired immune system and its potential to control a persistent viral infection.

As we describe the immune system in ever more exquisite detail, we might find that no matter how successful, this approach will not be sufficient to understand the spread of infectious agents, their susceptibility to vaccine therapy, and human disease resistance. Compared with the strict reductionism practiced as a means of characterizing most biological processes, I propose that the progression and outcome of disease-causing host-parasite interactions will be more clearly understood through a focus on disease ecology.