Stat5 opposes the transcription factor Tox and rewires exhausted CD8+ T cells toward durable effector-like states during chronic antigen exposure.

Rewiring exhausted CD8+ T (Tex) cells toward functional states remains a therapeutic challenge. Tex cells are epigenetically programmed by the transcription factor Tox. However, epigenetic remodeling occurs as Tex cells transition from progenitor (Texprog) to intermediate (Texint) and terminal (Texterm) subsets, suggesting development flexibility. We examined epigenetic transitions between Tex cell subsets and revealed a reciprocally antagonistic circuit between Stat5a and Tox. Stat5 directed Texint cell formation and re-instigated partial effector biology during this Texprog-to-Texint cell transition. Constitutive Stat5a activity antagonized Tox and rewired CD8+ T cells from exhaustion to a durable effector and/or natural killer (NK)-like state with superior anti-tumor potential. Temporal induction of Stat5 activity in Tex cells using an orthogonal IL-2:IL2Rß-pair fostered Texint cell accumulation, particularly upon PD-L1 blockade. Re-engaging Stat5 also partially reprogrammed the epigenetic landscape of exhaustion and restored polyfunctionality. These data highlight therapeutic opportunities of manipulating the IL-2-Stat5 axis to rewire Tex cells toward more durably protective states.

Feasibility and reliability of the pandemic-adapted online-onsite hybrid graduation OSCE in Japan.

Objective structured clinical examination (OSCE) is widely used to assess medical students' clinical skills. Virtual OSCEs were used in place of in-person OSCEs during the COVID-19 pandemic; however, their reliability is yet to be robustly analyzed. By applying generalizability (G) theory, this study aimed to evaluate the reliability of a hybrid OSCE, which admixed in-person and online methods, and gain insights into improving OSCEs' reliability. During the 2020-2021 hybrid OSCEs, one examinee, one rater, and a vinyl mannequin for physical examination participated onsite, and a standardized simulated patient (SP) for medical interviewing and another rater joined online in one virtual breakout room on an audiovisual conferencing system. G-coefficients and 95% confidence intervals of the borderline score, namely border zone (BZ), under the standard 6-station, 2-rater, and 6-item setting were calculated. G-coefficients of in-person (2017-2019) and hybrid OSCEs (2020-2021) under the standard setting were estimated to be 0.624, 0.770, 0.782, 0.759, and 0.823, respectively. The BZ scores were estimated to be 2.43-3.57, 2.55-3.45, 2.59-3.41, 2.59-3.41, and 2.51-3.49, respectively, in the score range from 1 to 6. Although hybrid OSCEs showed reliability comparable to in-person OSCEs, they need further improvement as a very high-stakes examination. In addition to increasing clinical vignettes, having more proficient online/on-demand raters and/or online SPs for medical interviews could improve the reliability of OSCEs. Reliability can also be ensured through supplementary examination and by increasing the number of online raters for a small number of students within the BZs.

Met receptor is essential for MAVS-mediated antiviral innate immunity in epithelial cells independent of its kinase activity.

Epithelial tissue is at the forefront of innate immunity, playing a crucial role in the recognition and elimination of pathogens. Met is a receptor tyrosine kinase that is necessary for epithelial cell survival, proliferation, and regeneration. Here, we showed that Met is essential for the induction of cytokine production by cytosolic nonself double-stranded RNA through retinoic acid-inducible gene-I-like receptors (RLRs) in epithelial cells. Surprisingly, the tyrosine kinase activity of Met was dispensable for promoting cytokine production. Rather, the intracellular carboxy terminus of Met interacted with mitochondrial antiviral-signaling protein (MAVS) in RLR-mediated signaling to directly promote MAVS signalosome formation. These studies revealed a kinase activity-independent function of Met in the promotion of antiviral innate immune responses, defining dual roles of Met in both regeneration and immune responses in the epithelium.

Leukocyte cell-derived chemotaxin 2 is an antiviral regulator acting through the proto-oncogene MET.

Retinoic acid-inducible gene (RIG)-I is an essential innate immune sensor that recognises pathogen RNAs and induces interferon (IFN) production. However, little is known about how host proteins regulate RIG-I activation. Here, we show that leukocyte cell-derived chemotaxin 2 (LECT2), a hepatokine and ligand of the MET receptor tyrosine kinase is an antiviral regulator that promotes the RIG-I-mediated innate immune response. Upon binding to MET, LECT2 induces the recruitment of the phosphatase PTP4A1 to MET and facilitates the dissociation and dephosphorylation of phosphorylated SHP2 from MET, thereby protecting RIG-I from SHP2/c-Cbl-mediated degradation. In vivo, LECT2 overexpression enhances RIG-I-dependent IFN production and inhibits lymphocytic choriomeningitis virus (LCMV) replication in the liver, whereas these changes are reversed in LECT2 knockout mice. Forced suppression of MET abolishes IFN production and antiviral activity in vitro and in vivo. Interestingly, hepatocyte growth factor (HGF), an original MET ligand, inhibits LECT2-mediated anti-viral signalling; conversely, LECT2-MET signalling competes with HGF-MET signalling. Our findings reveal previously unrecognized crosstalk between MET-mediated proliferation and innate immunity and suggest that targeting LECT2 may have therapeutic value in infectious diseases and cancer.

MicroRNA-29a attenuates CD8 T cell exhaustion and induces memory-like CD8 T cells during chronic infection.

CD8 T cells mediate protection against intracellular pathogens and tumors. However, persistent antigen during chronic infections or cancer leads to T cell exhaustion, suboptimal functionality, and reduced protective capacity. Despite considerable work interrogating the transcriptional regulation of exhausted CD8 T cells (TEX), the posttranscriptional control of TEX remains poorly understood. Here, we interrogated the role of microRNAs (miRs) in CD8 T cells responding to acutely resolved or chronic viral infection and identified miR-29a as a key regulator of TEX. Enforced expression of miR-29a improved CD8 T cell responses during chronic viral infection and antagonized exhaustion. miR-29a inhibited exhaustion-driving transcriptional pathways, including inflammatory and T cell receptor signaling, and regulated ribosomal biogenesis. As a result, miR-29a fostered a memory-like CD8 T cell differentiation state during chronic infection. Thus, we identify miR-29a as a key regulator of TEX and define mechanisms by which miR-29a can divert exhaustion toward a more beneficial memory-like CD8 T cell differentiation state.

Batf-mediated epigenetic control of effector CD8+ T cell differentiation.

The response of naive CD8+ T cells to their cognate antigen involves rapid and broad changes to gene expression that are coupled with extensive chromatin remodeling, but the mechanisms governing these changes are not fully understood. Here, we investigated how these changes depend on the basic leucine zipper ATF-like transcription factor Batf, which is essential for the early phases of the process. Through genome scale profiling, we characterized the role of Batf in chromatin organization at several levels, including the accessibility of key regulatory regions, the expression of their nearby genes, and the interactions that these regions form with each other and with key transcription factors. We identified a core network of transcription factors that cooperated with Batf, including Irf4, Runx3, and T-bet, as indicated by their colocalization with Batf and their binding in regions whose accessibility, interactions, and expression of nearby genes depend on Batf. We demonstrated the synergistic activity of this network by overexpressing the different combinations of these genes in fibroblasts. Batf and Irf4, but not Batf alone, were sufficient to increase accessibility and transcription of key loci, normally associated with T cell function. Addition of Runx3 and T-bet further contributed to fine-tuning of these changes and was essential for establishing chromatin loops characteristic of T cells. These data provide a resource for studying the epigenomic and transcriptomic landscape of effector differentiation of cytotoxic T cells and for investigating the interdependency between transcription factors and its effects on the epigenome and transcriptome of primary cells.

Transient Depletion of CD4+ Cells Induces Remodeling of the TCR Repertoire in Gastrointestinal Cancer.

Antibody-mediated transient depletion of CD4+ cells enhances the expansion of tumor-reactive CD8+ T cells and exhibits robust antitumor effects in preclinical and clinical studies. To investigate the clonal T-cell responses following transient CD4+ cell depletion in patients with cancer, we conducted a temporal analysis of the T-cell receptor (TCR) repertoire in the first-in-human clinical trial of IT1208, a defucosylated humanized monoclonal anti-CD4. Transient depletion of CD4+ cells promoted replacement of T-cell clones among CD4+ and CD8+ T cells in the blood. This replacement of the TCR repertoire was associated with the extent of CD4+ T-cell depletion and an increase in CD8+ T-cell count in the blood. Next, we focused on T-cell clones overlapping between the blood and tumor in order to track tumor-associated T-cell clones in the blood. The total frequency of blood-tumor overlapping clones tended to increase in patients receiving a depleting dose of anti-CD4, which was accompanied by the replacement of overlapping clones. The greater expansion of CD8+ overlapping clones was commonly observed in the patients who achieved tumor shrinkage. These results suggested that the clonal replacement of the TCR repertoire induced by transient CD4+ cell depletion was accompanied by the expansion of tumor-reactive T-cell clones that mediated antitumor responses. Our findings propose beneficial remodeling of the TCR repertoire following transient CD4+ cell depletion and provide novel insight into the antitumor effects of monoclonal anti-CD4 treatment in patients with cancer.See related Spotlight on p. 601.

In vivo CD8+ T cell CRISPR screening reveals control by Fli1 in infection and cancer.

Improving effector activity of antigen-specific T cells is a major goal in cancer immunotherapy. Despite the identification of several effector T cell (TEFF)-driving transcription factors (TFs), the transcriptional coordination of TEFF biology remains poorly understood. We developed an in vivo T cell CRISPR screening platform and identified a key mechanism restraining TEFF biology through the ETS family TF, Fli1. Genetic deletion of Fli1 enhanced TEFF responses without compromising memory or exhaustion precursors. Fli1 restrained TEFF lineage differentiation by binding to cis-regulatory elements of effector-associated genes. Loss of Fli1 increased chromatin accessibility at ETS:RUNX motifs, allowing more efficient Runx3-driven TEFF biology. CD8+ T cells lacking Fli1 provided substantially better protection against multiple infections and tumors. These data indicate that Fli1 safeguards the developing CD8+ T cell transcriptional landscape from excessive ETS:RUNX-driven TEFF cell differentiation. Moreover, genetic deletion of Fli1 improves TEFF differentiation and protective immunity in infections and cancer.

Inhibitory signaling sustains a distinct early memory CD8+ T cell precursor that is resistant to DNA damage.

The developmental origins of memory T cells remain incompletely understood. During the expansion phase of acute viral infection, we identified a distinct subset of virus-specific CD8+ T cells that possessed distinct characteristics including expression of CD62L, T cell factor 1 (TCF-1), and Eomesodermin; relative quiescence; expression of activation markers; and features of limited effector differentiation. These cells were a quantitatively minor subpopulation of the TCF-1+ pool and exhibited self-renewal, heightened DNA damage surveillance activity, and preferential long-term recall capacity. Despite features of memory and somewhat restrained proliferation during the expansion phase, this subset displayed evidence of stronger TCR signaling than other responding CD8+ T cells, coupled with elevated expression of multiple inhibitory receptors including programmed cell death 1 (PD-1), lymphocyte activating gene 3 (LAG-3), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), CD5, and CD160. Genetic ablation of PD-1 and LAG-3 compromised the formation of this CD62Lhi TCF-1+ subset and subsequent CD8+ T cell memory. Although central memory phenotype CD8+ T cells were formed in the absence of these cells, subsequent memory CD8+ T cell recall responses were compromised. Together, these results identify an important link between genome integrity maintenance and CD8+ T cell memory. Moreover, the data indicate a role for inhibitory receptors in preserving key memory CD8+ T cell precursors during initial activation and differentiation. Identification of this rare subpopulation within the memory CD8+ T cell precursor pool may help reconcile models of the developmental origin of long-term CD8+ T cell memory.

MCPIP1 reduces HBV-RNA by targeting its epsilon structure.

Hepatitis B virus (HBV) is the major causative factor of chronic viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. We previously demonstrated that a proinflammatory cytokine IL-1ß reduced the level of HBV RNA. However, the mechanism underlying IL-1ß-mediated viral RNA reduction remains incompletely understood. In this study, we report that immune regulator Monocyte chemotactic protein-1-induced protein 1 (MCPIP1) can reduce HBV RNA in hepatocytes. MCPIP1 expression level was higher in the liver tissue of HBV-infected patients and mice. Overexpression of MCPIP1 decreased HBV RNA, whereas ablating MCPIP1 in vitro enhanced HBV production. The domains responsible for RNase activity or oligomerization, were required for MCPIP1-mediated viral RNA reduction. The epsilon structure of HBV RNA was important for its antiviral activity and cleaved by MCPIP1 in the cell-free system. Lastly, knocking out MCPIP1 attenuated the anti-HBV effect of IL-1ß, suggesting that MCPIP1 is required for IL-1ß-mediated HBV RNA reduction. Overall, these results suggest that MCPIP1 may be involved in the antiviral effect downstream of IL-1ß.

Developmental Relationships of Four Exhausted CD8+ T Cell Subsets Reveals Underlying Transcriptional and Epigenetic Landscape Control Mechanisms.

CD8+ T cell exhaustion is a major barrier to current anti-cancer immunotherapies. Despite this, the developmental biology of exhausted CD8+ T cells (Tex) remains poorly defined, restraining improvement of strategies aimed at "re-invigorating" Tex cells. Here, we defined a four-cell-stage developmental framework for Tex cells. Two TCF1+ progenitor subsets were identified, one tissue restricted and quiescent and one more blood accessible, that gradually lost TCF1 as it divided and converted to a third intermediate Tex subset. This intermediate subset re-engaged some effector biology and increased upon PD-L1 blockade but ultimately converted into a fourth, terminally exhausted subset. By using transcriptional and epigenetic analyses, we identified the control mechanisms underlying subset transitions and defined a key interplay between TCF1, T-bet, and Tox in the process. These data reveal a four-stage developmental hierarchy for Tex cells and define the molecular, transcriptional, and epigenetic mechanisms that could provide opportunities to improve cancer immunotherapy.

Trib1 regulates T cell differentiation during chronic infection by restraining the effector program.

In chronic infections, the immune response fails to control virus, leading to persistent antigen stimulation and the progressive development of T cell exhaustion. T cell effector differentiation is poorly understood in the context of exhaustion, but targeting effector programs may provide new strategies for reinvigorating T cell function. We identified Tribbles pseudokinase 1 (Trib1) as a central regulator of antiviral T cell immunity, where loss of Trib1 led to a sustained enrichment of effector-like KLRG1+ T cells, enhanced function, and improved viral control. Single-cell profiling revealed that Trib1 restrains a population of KLRG1+ effector CD8 T cells that is transcriptionally distinct from exhausted cells. Mechanistically, we identified an interaction between Trib1 and the T cell receptor (TCR) signaling activator, MALT1, which disrupted MALT1 signaling complexes. These data identify Trib1 as a negative regulator of TCR signaling and downstream function, and reveal a link between Trib1 and effector versus exhausted T cell differentiation that can be targeted to improve antiviral immunity.

TCF-1-Centered Transcriptional Network Drives an Effector versus Exhausted CD8 T Cell-Fate Decision.

TCF-1 is a key transcription factor in progenitor exhausted CD8 T cells (Tex). Moreover, this Tex cell subset mediates responses to PD-1 checkpoint pathway blockade. However, the role of the transcription factor TCF-1 in early fate decisions and initial generation of Tex cells is unclear. Single-cell RNA sequencing (scRNA-seq) and lineage tracing identified a TCF-1+Ly108+PD-1+ CD8 T cell population that seeds development of mature Tex cells early during chronic infection. TCF-1 mediated the bifurcation between divergent fates, repressing development of terminal KLRG1Hi effectors while fostering KLRG1Lo Tex precursor cells, and PD-1 stabilized this TCF-1+ Tex precursor cell pool. TCF-1 mediated a T-bet-to-Eomes transcription factor transition in Tex precursors by promoting Eomes expression and drove c-Myb expression that controlled Bcl-2 and survival. These data define a role for TCF-1 in early-fate-bifurcation-driving Tex precursor cells and also identify PD-1 as a protector of this early TCF-1 subset.

CXCR6 regulates localization of tissue-resident memory CD8 T cells to the airways.

Resident memory T cells (TRM cells) are an important first-line defense against respiratory pathogens, but the unique contributions of lung TRM cell populations to protective immunity and the factors that govern their localization to different compartments of the lung are not well understood. Here, we show that airway and interstitial TRM cells have distinct effector functions and that CXCR6 controls the partitioning of TRM cells within the lung by recruiting CD8 TRM cells to the airways. The absence of CXCR6 significantly decreases airway CD8 TRM cells due to altered trafficking of CXCR6-/- cells within the lung, and not decreased survival in the airways. CXCL16, the ligand for CXCR6, is localized primarily at the respiratory epithelium, and mice lacking CXCL16 also had decreased CD8 TRM cells in the airways. Finally, blocking CXCL16 inhibited the steady-state maintenance of airway TRM cells. Thus, the CXCR6/CXCL16 signaling axis controls the localization of TRM cells to different compartments of the lung and maintains airway TRM cells.

Different antiviral activities of natural APOBEC3C, APOBEC3G, and APOBEC3H variants against hepatitis B virus.

Some APOBEC3 family members have antiviral activity against retroviruses and DNA viruses. Hepatitis B virus (HBV) is a DNA virus that is the major causative factor of severe liver diseases such as cirrhosis and hepatocellular carcinoma. To determine whether APOBEC3 variants in humans have different anti-HBV activities, we evaluated natural variants of APOBEC3C, APOBEC3G, and APOBEC3H using an HBV-replicating cell culture model. Our data demonstrate that the APOBEC3C variant S188I had increased restriction activity and hypermutation frequency against HBV DNA. In contrast, the APOBEC3G variant H186R did not alter the anti-HBV and hypermutation activities. Among APOBEC3H polymorphisms (hap I-VII) and splicing variants (SV-200, SV-183, SV-182, and SV-154), hap II SV-183 showed the strongest restriction activity. These data suggest that the genetic variations in APOBEC3 genes may affect the efficiency of HBV elimination in humans.

CD8+ T cell exhaustion.

CD8+ T cells are important for the protective immunity against intracellular pathogens and tumor. In the case of chronic infection or cancer, CD8+ T cells are exposed to persistent antigen and/or inflammatory signals. This excessive amount of signals often leads CD8+ T cells to gradual deterioration of T cell function, a state called "exhaustion." Exhausted T cells are characterized by progressive loss of effector functions (cytokine production and killing function), expression of multiple inhibitory receptors (such as PD-1 and LAG3), dysregulated metabolism, poor memory recall response, and homeostatic proliferation. These altered functions are closely related with altered transcriptional program and epigenetic landscape that clearly distinguish exhausted T cells from normal effector and memory T cells. T cell exhaustion is often associated with inefficient control of persisting infections and cancers, but re-invigoration of exhausted T cells with inhibitory receptor blockade can promote improved immunity and disease outcome. Accumulating evidences support the therapeutic potential of targeting exhausted T cells. However, exhausted T cells comprise heterogenous cell population with distinct responsiveness to intervention. Understanding molecular mechanism of T cell exhaustion is essential to establish rational immunotherapeutic interventions.

Long-Term Persistence of Exhausted CD8 T Cells in Chronic Infection Is Regulated by MicroRNA-155.

Persistent viral infections and tumors drive development of exhausted T (TEX) cells. In these settings, TEX cells establish an important host-pathogen or host-tumor stalemate. However, TEX cells erode over time, leading to loss of pathogen or cancer containment. We identified microRNA (miR)-155 as a key regulator of sustained TEX cell responses during chronic lymphocytic choriomeningitis virus (LCMV) infection. Genetic deficiency of miR-155 ablated CD8 T cell responses during chronic infection. Conversely, enhanced miR-155 expression promoted expansion and long-term persistence of TEX cells. However, rather than strictly antagonizing exhaustion, miR-155 promoted a terminal TEX cell subset. Transcriptional profiling identified coordinated control of cell signaling and transcription factor pathways, including the key AP-1 family member Fosl2. Overexpression of Fosl2 reversed the miR-155 effects, identifying a link between miR-155 and the AP-1 transcriptional program in regulating TEX cells. Thus, we identify a mechanism of miR-155 regulation of TEX cells and a key role for Fosl2 in T cell exhaustion.

Generation of tumor antigen-specific murine CD8+ T cells with enhanced anti-tumor activity via highly efficient CRISPR/Cas9 genome editing.

Immunotherapies have led to the successful development of novel therapies for cancer. However, there is increasing concern regarding the adverse effects caused by non-tumor-specific immune responses. Here, we report an effective strategy to generate high-avidity tumor-antigen-specific CTLs, using Cas9/single-guide RNA (sgRNA) ribonucleoprotein (RNP) delivery. As a proof-of-principle demonstration, we selected the gp100 melanoma-associated tumor antigen, and cloned the gp100-specific high-avidity TCR from gp100-immunized mice. To enable rapid structural dissection of the TCR, we developed a 3D protein structure modeling system for the TCR/antigen-major histocompatibility complex (pMHC) interaction. Combining these technologies, we efficiently generated gp100-specific PD-1(-) CD8+ T cells, and demonstrated that the genetically engineered CD8+ T cells have high avidity against melanoma cells both in vitro and in vivo. Our methodology offers computational prediction of the TCR response, and enables efficient generation of tumor antigen-specific CD8+ T cells that can neutralize tumor-induced immune suppression leading to a potentially powerful cancer therapeutic.

Lineage-Determining Transcription Factor TCF-1 Initiates the Epigenetic Identity of T Cells.

T cell development is orchestrated by transcription factors that regulate the expression of genes initially buried within inaccessible chromatin, but the transcription factors that establish the regulatory landscape of the T cell lineage remain unknown. Profiling chromatin accessibility at eight stages of T cell development revealed the selective enrichment of TCF-1 at genomic regions that became accessible at the earliest stages of development. TCF-1 was further required for the accessibility of these regulatory elements and at the single-cell level, it dictated a coordinate opening of chromatin in T cells. TCF-1 expression in fibroblasts generated de novo chromatin accessibility even at chromatin regions with repressive marks, inducing the expression of T cell-restricted genes. These results indicate that a mechanism by which TCF-1 controls T cell fate is through its widespread ability to target silent chromatin and establish the epigenetic identity of T cells.