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Clinical evaluation of immune reconstitution and health status during HIV-1 infection and anti-retroviral therapy (ART) is largely based on CD4+ T cell counts and viral load, measures that fail to take into account the CD8+ T cell subset, known to show features of accelerated aging in HIV disease. Here, we compare adenosine deaminase (ADA), glucose uptake receptor 1 (GLUT1), and leucine-rich repeat neuronal 3 (LRRN3) to CD38 expression and telomerase activity, two strong predictors of HIV disease progression. Our analysis revealed that reduced ADA, telomerase activity and LRRN3 gene expression were significantly associated with high CD38 and HLA-DR in CD8+ T cells, with % ADA+ cells being the most robust predictor of CD8+ T cell activation. Our results suggest that ADA, LRRN3 and telomerase activity in CD8+ T cells may serve as novel, clinically relevant biomarkers of immune status in HIV-1 infection, specifically by demonstrating the degree to which CD8+ T cells have progressed to the end stage of replicative senescence. Since chronological aging itself leads to the accumulation of senescent CD8+ T cells, the prolonged survival and resultant increased age of the HIV+ population may synergize with the chronic immune activation to exacerbate both immune decline and age-associated pathologies. The identification and future validation of these new biomarkers may lead to fresh immune-based HIV treatments.

CD28 costimulatory signal transduction in T lymphocytes is essential for optimal telomerase activity, stabilization of cytokine mRNAs, and glucose metabolism. During aging and chronic infection with HIV-1, there are increased proportions of CD8 T lymphocytes that lack CD28 expression and show additional features of replicative senescence. Moreover, the abundance of these cells correlates with decreased vaccine responsiveness, early mortality in the very old, and accelerated HIV disease progression. Here, we show that sustained expression of CD28, via gene transduction, retards the process of replicative senescence, as evidenced by enhanced telomerase activity, increased overall proliferative potential, and reduced secretion of pro-inflammatory cytokines. Nevertheless, the transduced cultures eventually do reach senescence, which is associated with increased CTLA-4 gene expression and a loss of CD28 cell surface expression. These findings further elucidate the central role of CD28 in the replicative senescence program, and may ultimately lead to novel therapies for diseases associated with replicative senescence.

Identifying molecular mechanisms of exhausted CD8 T cells (Tex) is a key goal of improving immunotherapy of cancer and other diseases. However, high-throughput interrogation of in vivo Tex can be costly and inefficient. In vitro models of Tex are easily customizable and quickly generate high cellular yield, enabling CRISPR screening and other high-throughput assays. We established an in vitro model of chronic stimulation and benchmarked key phenotypic, functional, transcriptional, and epigenetic features against bona fide in vivo Tex. We leveraged this model of in vitro chronic stimulation in combination with CRISPR screening to identify transcriptional regulators of T cell exhaustion. This approach identified several transcription factors, including BHLHE40. In vitro and in vivo validation defined a role for BHLHE40 in regulating a key differentiation checkpoint between progenitor and intermediate Tex subsets. By developing and benchmarking an in vitro model of Tex, then applying high-throughput CRISPR screening, we demonstrate the utility of mechanistically annotated in vitro models of Tex. Editor's summary: T cell exhaustion represents a distinct cellular state that contributes to immune dysfunction during chronic viral infections and cancer. To overcome limitations of in vivo models, Wu et al. developed and benchmarked an in vitro platform of T cell exhaustion. CD8+ T cells were cultured with dendritic cells repeatedly pulsed with cognate peptide and IL-2. This system reproduced the functional, transcriptomic, and epigenetic features of exhausted T cells generated during chronic lymphocytic choriomeningitis virus infection in mice. Using a pooled CRISPR screen targeting 120 transcription factors, they identified BHLHE40 as a regulator of T cell exhaustion, and genetic silencing of BHLHE40 prevented the development of more differentiated exhausted T cell subsets. Together, these findings provide a scalable and controlled platform for interrogating the biology underlying T cell exhaustion. —Claire Olingy [ABSTRACT FROM AUTHOR]

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibited higher basal levels of activation measured by P-selectin surface expression and had poor functional reserve upon in vitro stimulation. To investigate this question in more detail, we developed an assay to assess the capacity of plasma from COVID-19 patients to activate platelets from healthy donors. Platelet activation was a common feature of plasma from COVID-19 patients and correlated with key measures of clinical outcome including kidney and liver injury, and APACHEIII scores. Further, we identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions. These data identified these potentially actionable pathways as central for platelet activation and/or vascular complications and clinical outcomes in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect. [ABSTRACT FROM AUTHOR]

Pediatric versus adult COVID-19: Some severe pediatric patients with COVID-19 develop multisystem inflammatory syndrome in children (MIS-C), which associates with vascular damage. However, the immune correlates of pediatric MIS-C are not well defined. Vella et al. used multipanel flow cytometry of peripheral blood mononuclear cells from pediatric patients with MIS-C and compared them with adults with severe COVID-19. Many immunologic features were similar between MIS-C and severely ill adult patients with COVID-19, yet there was a significant increase in the activation of vascular patrolling CX3CR1+ CD8+ T cells, especially in children who needed vasoactive medication. As pediatric patients with MIS-C improved, these CX3CR1+ CD8+ T cells decreased. Thus, CX3CR1+ CD8+ T cells seem to correlate with pediatric MIS-C, potentially contributing to the vascular damage associated with the disease. Pediatric coronavirus disease 2019 (COVID-19) after SARS-CoV-2 infection is associated with fewer hospitalizations and often milder disease than in adults. A subset of children, however, present with multisystem inflammatory syndrome in children (MIS-C) that can lead to vascular complications and shock, but rarely death. The immune features of MIS-C compared with pediatric COVID-19 or adult disease remain poorly understood. We analyzed peripheral blood immune responses in hospitalized SARS-CoV-2–infected pediatric patients (pediatric COVID-19) and patients with MIS-C. Patients with MIS-C had patterns of T cell–biased lymphopenia and T cell activation similar to severely ill adults, and all patients with MIS-C had SARS-CoV-2 spike–specific antibodies at admission. A distinct feature of patients with MIS-C was robust activation of vascular patrolling CX3CR1+ CD8+ T cells that correlated with the use of vasoactive medication. Last, whereas pediatric COVID-19 patients with acute respiratory distress syndrome had sustained immune activation, patients with MIS-C displayed clinical improvement over time, concomitant with decreasing immune activation. Thus, non–MIS-C versus MIS-C SARS-CoV-2–associated illnesses are characterized by divergent immune signatures that are temporally distinct from one another and implicate CD8+ T cells in the clinical presentation and trajectory of MIS-C. [ABSTRACT FROM AUTHOR]

Exhausted CD8+ T (Tex) cells in chronic infections and cancer have limited effector function, high co-expression of inhibitory receptors and extensive transcriptional changes compared with effector (Teff) or memory (Tmem) CD8+ T cells. Tex cells are important clinical targets of checkpoint blockade and other immunotherapies. Epigenetically, Tex cells are a distinct immune subset, with a unique chromatin landscape compared with Teff and Tmem cells. However, the mechanisms that govern the transcriptional and epigenetic development of Tex cells remain unknown. Here we identify the HMG-box transcription factor TOX as a central regulator of Tex cells in mice. TOX is largely dispensable for the formation of Teff and Tmem cells, but it is critical for exhaustion: in the absence of TOX, Tex cells do not form. TOX is induced by calcineurin and NFAT2, and operates in a feed-forward loop in which it becomes calcineurin-independent and sustained in Tex cells. Robust expression of TOX therefore results in commitment to Tex cells by translating persistent stimulation into a distinct Tex cell transcriptional and epigenetic developmental program. The transcription factor TOX is a central regulator of the transcriptional and epigenetic development of exhausted T cells. [ABSTRACT FROM AUTHOR]

Significantly higher levels of plasma CXCL13 [chemokine (C-X-Cmotif) ligand 13] were associated with the generation of broadly neutralizing antibodies (bnAbs) against HIV in a large longitudinal cohort of HIV-infected individuals. Germinal centers (GCs) perform the remarkable task of optimizing B-cell Ab responses. GCs are required for almost all B-cell receptor affinity maturation and will be a critical parameter to monitor if HIV bnAbs are to be induced by vaccination. However, lymphoid tissue is rarely available from immunized humans,making themonitoring of GC activity by direct assessment of GC B cells and germinal center CD4+ T follicular helper (GC Tfh) cells problematic. The CXCL13-CXCR5 [chemokine (C-X-C motif) receptor 5] chemokine axis plays a central role in organizing both B-cell follicles and GCs. Because GC Tfh cells can produce CXCL13, we explored the potential use of CXCL13 as a blood biomarker to indicate GC activity. In a series of studies, we found that plasma CXCL13 levels correlated with GC activity in draining lymph nodes of immunized mice, immunized macaques, and HIV-infected humans. Furthermore, plasma CXCL13 levels in immunized humans correlated with the magnitude of Ab responses and the frequency of ICOS+ (inducible T-cell costimulator) Tfh-like cells in blood. Together, these findings support the potential use of CXCL13 as a plasma biomarker of GC activity in human vaccine trials and other clinical settings. [ABSTRACT FROM AUTHOR]

Improving effector activity of antigen-specific T cells is a major goal in cancer immunotherapy. Despite the identification of several effector T cell (T EFF )-driving transcription factors (TFs), the transcriptional coordination of T EFF biology remains poorly understood. We developed an in vivo T cell CRISPR screening platform and identified a key mechanism restraining T EFF biology through the ETS family TF, Fli1. Genetic deletion of Fli1 enhanced T EFF responses without compromising memory or exhaustion precursors. Fli1 restrained T EFF 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 T EFF 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 T EFF cell differentiation. Moreover, genetic deletion of Fli1 improves T EFF differentiation and protective immunity in infections and cancer., Competing Interests: Declaration of interests E.J.W. has consulting agreements with and/or is on the scientific advisory board for Merck, Elstar, Janssen, Related Sciences, Synthekine and Surface Oncology. E.J.W. is a founder of Surface Oncology and Arsenal Biosciences. E.J.W. has a patent licensing agreement on the PD-1 pathway with Roche/Genentech. O.K. is an employee and shareholder of Arsenal Biosciences., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Pediatric COVID-19 following SARS-CoV-2 infection is associated with fewer hospitalizations and often milder disease than in adults. A subset of children, however, present with Multisystem Inflammatory Syndrome in Children (MIS-C) that can lead to vascular complications and shock, but rarely death. The immune features of MIS-C compared to pediatric COVID-19 or adult disease remain poorly understood. We analyzed peripheral blood immune responses in hospitalized SARS-CoV-2 infected pediatric patients (pediatric COVID-19) and patients with MIS-C. MIS-C patients had patterns of T cell-biased lymphopenia and T cell activation similar to severely ill adults, and all patients with MIS-C had SARS-CoV-2 spike-specific antibodies at admission. A distinct feature of MIS-C patients was robust activation of vascular patrolling CX3CR1+ CD8+ T cells that correlated with the use of vasoactive medication. Finally, whereas pediatric COVID-19 patients with acute respiratory distress syndrome (ARDS) had sustained immune activation, MIS-C patients displayed clinical improvement over time, concomitant with decreasing immune activation. Thus, non-MIS-C versus MIS-C SARS-CoV-2 associated illnesses are characterized by divergent immune signatures that are temporally distinct from one another and implicate CD8+ T cells in the clinical presentation and trajectory of MIS-C., (Copyright © 2021, American Association for the Advancement of Science.)

Expanded populations of CD8(+) T lymphocytes lacking CD28 expression are associated with a variety of deleterious clinical outcomes, including early mortality in the elderly, more rapid progression to AIDS, cardiovascular disease, and enhanced tumor cell growth. In cell culture, irreversible loss of CD28 expression correlates with increased production of TNF-alpha as CD8(+) T cells are driven to the nonproliferative end stage of replicative senescence by multiple rounds of Ag-driven cell division. Interestingly, in patients with rheumatoid arthritis, inhibition or neutralization of TNF-alpha reduces the proportion of T cells lacking CD28 in the disease joints, consistent with studies showing a direct involvement of this cytokine in CD28 gene transcription. Here, we show that modulation of TNF-alpha levels in long-term cultures of human CD8(+) T lymphocytes, by chronic exposure either to a neutralizing Ab or to an inhibitor of the TNF-alpha receptor-1, increases proliferative potential, delays loss of CD28 expression, retards cytokine profile changes, and enhances telomerase activity. We also show that constitutive caspase-3, one of the downstream effectors of TNF-alphaR1 binding, increases in parallel with the loss of CD28 in long-term cultures, but this effect is blunted in the presence of the TNF-alpha inhibitors. Consistent with the in vitro culture data, CD8(+)CD28(-) T lymphocytes tested immediately ex vivo also show significantly higher levels of caspase-3 compared with their CD28(+) counterparts. These findings help elucidate the complex nature of CD28 gene regulation, and may ultimately lead to novel therapeutic approaches for diseases associated with increased proportions of CD28(-) T lymphocytes.