STING controls T cell memory fitness during infection through T cell-intrinsic and IDO-dependent mechanisms.

Stimulator of interferon genes (STING) signaling has been extensively studied in inflammatory diseases and cancer, while its role in T cell responses to infection is unclear. Using Listeria monocytogenes strains engineered to induce different levels of c-di-AMP, we found that high STING signals impaired T cell memory upon infection via increased Bim levels and apoptosis. Unexpectedly, reduction of TCR signal strength or T cell-STING expression decreased Bim expression, T cell apoptosis, and recovered T cell memory. We found that TCR signal intensity coupled STING signal strength to the unfolded protein response (UPR) and T cell survival. Under strong STING signaling, Indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibition also reduced apoptosis and led to a recovery of T cell memory in STING sufficient CD8 T cells. Thus, STING signaling regulates CD8 T cell memory fitness through both cell-intrinsic and extrinsic mechanisms. These studies provide insight into how IDO and STING therapies could improve long-term T cell protective immunity.

Type II Cytokines Fine-Tune Thymic T Cell Selection to Offset Murine Central Nervous System Autoimmunity.

Early thymic progenitors (ETPs) are bone marrow-derived hematopoietic stem cells that remain multipotent and give rise to a variety of lineage-specific cells. Recently, we discovered a subset of murine ETPs that expresses the IL-4Rα/IL-13Rα1 heteroreceptor (HR) and commits only to the myeloid lineage. This is because IL-4/IL-13 signaling through the HR inhibits their T cell potential and enacts commitment of HR+ETPs to thymic resident CD11c+CD8α+ dendritic cells (DCs). In this study, we discovered that HR+-ETP-derived DCs function as APCs in the thymus and promote deletion of myelin-reactive T cells. Furthermore, this negative T cell selection function of HR+-ETP-derived DCs sustains protection against experimental allergic encephalomyelitis, a mouse model for human multiple sclerosis. These findings, while shedding light on the intricacies underlying ETP lineage commitment, reveal a novel, to our knowledge, function by which IL-4 and IL-13 cytokines condition thymic microenvironment to rheostat T cell selection and fine-tune central tolerance.

NFκB-Pim-1-Eomesodermin axis is critical for maintaining CD8 T-cell memory quality.

T-cell memory is critical for long-term immunity. However, the factors involved in maintaining the persistence, function, and phenotype of the memory pool are undefined. Eomesodermin (Eomes) is required for the establishment of the memory pool. Here, we show that in T cells transitioning to memory, the expression of high levels of Eomes is not constitutive but rather requires a continuum of cell-intrinsic NFκB signaling. Failure to maintain NFκB signals after the peak of the response led to impaired Eomes expression and a defect in the maintenance of CD8 T-cell memory. Strikingly, we found that antigen receptor [T-cell receptor (TCR)] signaling regulates this process through expression of the NFκB-dependent kinase proviral integration site for Moloney murine leukemia virus-1 (PIM-1), which in turn regulates NFκB and Eomes. T cells defective in TCR-dependent NFκB signaling were impaired in late expression of Pim-1, Eomes, and CD8 memory. These defects were rescued when TCR-dependent NFκB signaling was restored. We also found that NFκB-Pim-1 signals were required at memory to maintain memory CD8 T-cell longevity, effector function, and Eomes expression. Hence, an NFκB-Pim-1-Eomes axis regulates Eomes levels to maintain memory fitness.

POSH Regulates CD4+ T Cell Differentiation and Survival.

The scaffold molecule POSH is crucial for the regulation of proliferation and effector function in CD8(+) T cells. However, its role in CD4(+) T cells is not known. In this study, we found that disruption of the POSH scaffold complex established a transcriptional profile that strongly skewed differentiation toward Th2, led to decreased survival, and had no effect on cell cycle entry. This is in stark contrast to CD8(+) T cells in which POSH regulates cell cycle and does not affect survival. Disruption of POSH in CD4(+) T cells resulted in the loss of Tak1-dependent activation of JNK1/2 and Tak1-mediated survival. However, in CD8(+) T cells, POSH regulates only JNK1. Remarkably, each type of T cell had a unique composition of the POSH scaffold complex and distinct posttranslational modifications of POSH. These data indicate that the mechanism that regulates POSH function in CD4(+) T cells is different from CD8(+) T cells. All together, these data strongly suggest that POSH is essential for the integration of cell-type-specific signals that regulate the differentiation, survival, and function of T cells.

IL-12 Signals through the TCR To Support CD8 Innate Immune Responses.

CD8 T cells must integrate antigenic and inflammatory signals to differentiate into efficient effector and memory T cells able to protect us from infections. The mechanisms by which TCR signaling and proinflammatory cytokine receptor signaling cooperate in these processes are poorly defined. In this study, we show that IL-12 and other proinflammatory cytokines transduce signals through the TCR signalosome in a manner that requires Fyn activity and self-peptide-MHC (self-pMHC) interactions. This mechanism is crucial for CD8 innate T cell functions. Loss of Fyn activity or blockade of self-pMHC interactions severely impaired CD8 T cell IFN-γ and NKG2D expression, proliferation, and cytotoxicity upon cytokine-mediated bystander activation. Most importantly, in the absence of self-pMHC interactions, CD8 memory T cells fail to undergo bystander activation upon an unrelated infection. Thus, CD8 T cell bystander activation, although independent of cognate Ag, still requires self-pMHC and TCR signaling.

Cutting edge: The signals for the generation of T cell memory are qualitatively different depending on TCR ligand strength.

CD8 T cell memory critically contributes to long-term immunity. Both low- and high-affinity TCR signals are able to support the differentiation of memory CD8 T cells. However, it is unclear whether the requirements for memory development change when TCR signal strength is altered. To gain further insight into this question, we used a TCRß transmembrane domain mutant model that is defective in the generation of memory in response to high-affinity ligands. Surprisingly, lowering TCR signal strength, by stimulation with low-affinity ligands, resulted in normal memory development. Restoration of memory correlated with recovery of TCR-dependent NF-κB signaling. Thus, these data provide novel evidence that the requirements for memory are qualitatively different depending on TCR signal strength.

The POSH/JIP-1 scaffold network regulates TCR-mediated JNK1 signals and effector function in CD8(+) T cells.

Signals from the T-cell recognition of antigen program effector functions are necessary to clear infections and tumors. The JNK pathway is critically important in regulating this process. In T lymphocytes, JNK1 and JNK2 have distinct functions depending on their maturation state and cell-type. However, the mechanisms that regulate their isoform-specific activity and function are still unclear. Here, we identify plenty of SH3 (POSH) and JNK-interacting protein 1 (JIP-1) as a multiprotein scaffold network for TCR-mediated JNK1 activation in CD8(+) T cells. Disruption of the POSH/JIP-1 complex led to profound defects in the activation of JNK1, as well as deficient activation or induction of the transcription factors c-Jun, T-bet, and Eomesodermin. Furthermore, disruption of the POSH/JIP complex in CD8(+) T cells resulted in impaired proliferation, decreased cytokine expression, and the inability to control tumors. Collectively, these data identify a mechanism for the specific regulation of TCR-dependent JNK1 activation and function that is key for CD8(+) T-cell responses.

Characterizing Osteochondral Allograft Biomechanics for Optimizing Transplant Success: A Systematic Review.

Osteochondral allograft (OCA) transplantation has been largely successful in treating symptomatic articular cartilage lesions; however, treatment failures persist. While OCA biomechanics have been consistently cited as mechanisms of treatment failure, the relationships among mechanical and biological variables that contribute to success after OCA transplantation have yet to be fully characterized. The purpose of this systematic review was to synthesize the clinically relevant peer-reviewed evidence targeting the biomechanics of OCAs and the impact on graft integration and functional survival toward developing and implementing strategies for improving patient outcomes. The Cochrane Central Register of Controlled Trials, the Cochrane Database of Systematic Reviews, MEDLINE, PubMed, Cumulative Index to Nursing and Allied Health (CINAHL), Google Scholar, and EMBASE were searched to identify articles for systematic review. This review of relevant peer-reviewed literature provided evidence that the biomechanics related to OCA transplantation in the knee have direct and indirect effects on functional graft survival and patient outcomes. The evidence suggests that biomechanical variables can be optimized further to enhance benefits and mitigate detrimental effects. Each of these modifiable variables should be considered regarding indications, patient selection criteria, graft preservation methodology, graft preparation, transplantation, fixation techniques, and prescribed postoperative restriction and rehabilitation protocols. Criteria, methods, techniques, and protocols should target OCA quality (chondrocyte viability, extracellular matrix integrity, material properties), favorable patient and joint characteristics, rigid fixation with protected loading, and innovative ways to foster rapid and complete OCA cartilage and bone integration to optimize outcomes for OCA transplant patients.

Cell-Mediated Immune Responses May Play Roles in Osteochondral Allograft Transplantation Osteointegration Failures.

Prolonged and incomplete osteochondral allograft (OCA) osteointegration is consistently cited as a major mechanism for OCA treatment failure. Subrejection immune responses may play roles in this mode of failure. Preimplantation OCA preparation techniques, including subchondral bone drilling, thorough irrigation, and autogenous bone marrow aspirate concentrate saturation, may dampen immune responses and improve OCA osteointegration. This study sought to further characterize potential immune system contributions to OCA transplantation treatment failures by analyzing donor-recipient ABO and Rh-factor mismatches and histological and immunohistochemical assessments of transplanted OCA tissues recovered from revision surgeries. Using a dedicated registry, OCA transplant recipients with documented treatment failures who met inclusion criteria (n = 33) as well as age-, body mass index-, and joint-matched patients with successful outcomes (n = 70) were analyzed to compare matched cohorts of patients with successful versus failed OCA transplantation outcomes. Tissues recovered from 18 failed OCA transplants and portions of 7 nonimplanted OCA controls were further analyzed to provide contributing evidence for potential immune response mechanisms. For patients analyzed, no statistically significant differences in proportions for treatment success versus failure based on mismatches for ABO type, Rh factor, or both were noted. Further, no statistically significant differences in proportions for histological immune response presence or absence based on mismatches for ABO type, Rh factor, or both were noted. Twelve (67%) of the failed OCA tissues contained lymphocyte aggregations in the subchondral bone, which were comprised of combinations of CD3 + , CD4 + , CD8 + , and CD20+ lymphocytes. The mechanisms of failure for these 12 OCA transplants involved insufficient OCA osteointegration. Results of this study suggest that T- and B-cell-mediated subrejection immune responses may play roles in OCA transplant treatment failures independent of donor-recipient blood type mismatch effects.

Rigid crosslinking of the CD3 complex leads to superior T cell stimulation.

Functionally bivalent non-covalent Fab dimers (Bi-Fabs) specific for the TCR/CD3 complex promote CD3 signaling on T cells. While comparing functional responses to stimulation with Bi-Fab, F(ab')2 or mAb specific for the same CD3 epitope, we observed fratricide requiring anti-CD3 bridging of adjacent T cells. Surprisingly, anti-CD3 Bi-Fab ranked first in fratricide potency, followed by anti-CD3 F(ab')2 and anti-CD3 mAb. Low resolution structural studies revealed anti-CD3 Bi-Fabs and F(ab')2 adopt similar global shapes with CD3-binding sites oriented outward. However, under molecular dynamic simulations, anti-CD3 Bi-Fabs crosslinked CD3 more rigidly than F(ab')2. Furthermore, molecular modelling of Bi-Fab and F(ab')2 binding to CD3 predicted crosslinking of T cell antigen receptors located in opposing plasma membrane domains, a feature fitting with T cell fratricide observed. Thus, increasing rigidity of Fab-CD3 crosslinking between opposing effector-target pairs may result in stronger T cell effector function. These findings could guide improving clinical performance of bi-specific anti-CD3 drugs.

NF-kB's contribution to B cell fate decisions.

NF-κB signaling is essential to an effective innate and adaptive immune response. Many immune-specific functional and developmental outcomes depend in large on NF-κB. The formidable task of sorting out the mechanisms behind the regulation and outcome of NF-κB signaling remains an important area of immunology research. Here we briefly discuss the role of NF-κB in regulating cell fate decisions at various times in the path of B cell development, activation, and the generation of long-term humoral immunity.

In Vitro Analysis of Thymocyte Signaling.

When a developing thymocyte expresses a TCR, it is subjected to numerous interactions with self-peptide/MHC complexes that determine its fate. These include death by neglect, negative selection (apoptosis and lineage deviation), positive selection, and lineage commitment. Identifying signals that govern these unique cell fates requires the ability to assess the activity, level of expression, subcellular location, and molecular associations between numerous proteins within the developing T cell. Given the unique, temporal, and developmental changes that occur during development, isolating and analyzing small populations of thymocytes are necessary to get a complete picture of the development process. Thus, this chapter describes methods designed to analyze thymocyte signaling under various types of peptide-based stimulation in vitro.

Fetal Thymic Organ Culture and Negative Selection.

Negative selection removes potentially harmful T cell precursors from the conventional T cell pool. This process can involve the induction of apoptosis, anergy, receptor editing, or deviation into a regulatory T cell lineage. As such, this process is essential for the health of an organism through its contribution to central and peripheral tolerance. While a great deal is known about the process, the precise mechanisms that regulate these various forms of negative selection are not clear. Numerous models exist with the potential to address these questions in vitro and in vivo. This chapter describes fetal thymic organ culture methods designed to analyze the signals that determine these unique cell fates.

NFκB signaling in T cell memory.

Memory T cells play an essential role in protecting against infectious diseases and cancer and contribute to autoimmunity and transplant rejection. Understanding how they are generated and maintained in the context of infection or vaccination holds promise to improve current immune-based therapies. At the beginning of any immune response, naïve T cells are activated and differentiate into cells with effector function capabilities. In the context of infection, most of these cells die once the pathogenic antigen has been cleared. Only a few of them persist and differentiate into memory T cells. These memory T cells are essential to host immunity because they are long-lived and can perform effector functions immediately upon re-infection. How a cell becomes a memory T cell and continues being one for months and even years past the initial infection is still not fully understood. Recent reviews have thoroughly discussed the transcriptional, epigenomic, and metabolic mechanisms that govern T cell memory differentiation. Yet much less is known of how signaling pathways that are common circuitries of multiple environmental signals regulate T cell outcome and, precisely, T cell memory. The function of the NFκB signaling system is perhaps best understood in innate cells. Recent findings suggest that NFκB signaling plays an essential and unique role in generating and maintaining CD8 T cell memory. This review aims to summarize these findings and discuss the remaining questions in the field.

IKK2/NFkB signaling controls lung resident CD8+ T cell memory during influenza infection.

CD8+ T cell tissue resident memory (TRM) cells are especially suited to control pathogen spread at mucosal sites. However, their maintenance in lung is short-lived. TCR-dependent NFkB signaling is crucial for T cell memory but how and when NFkB signaling modulates tissue resident and circulating T cell memory during the immune response is unknown. Here, we find that enhancing NFkB signaling in T cells once memory to influenza is established, increases pro-survival Bcl-2 and CD122 levels thus boosting lung CD8+ TRM maintenance. By contrast, enhancing NFkB signals during the contraction phase of the response leads to a defect in CD8+ TRM differentiation without impairing recirculating memory subsets. Specifically, inducible activation of NFkB via constitutive active IKK2 or TNF interferes with TGFß signaling, resulting in defects of lung CD8+ TRM imprinting molecules CD69, CD103, Runx3 and Eomes. Conversely, inhibiting NFkB signals not only recovers but improves the transcriptional signature and generation of lung CD8+ TRM. Thus, NFkB signaling is a critical regulator of tissue resident memory, whose levels can be tuned at specific times during infection to boost lung CD8+ TRM.

Thymic selection threshold defined by compartmentalization of Ras/MAPK signalling.

A healthy individual can mount an immune response to exogenous pathogens while avoiding an autoimmune attack on normal tissues. The ability to distinguish between self and non-self is called 'immunological tolerance' and, for T lymphocytes, involves the generation of a diverse pool of functional T cells through positive selection and the removal of overtly self-reactive thymocytes by negative selection during T-cell ontogeny. To elucidate how thymocytes arrive at these cell fate decisions, here we have identified ligands that define an extremely narrow gap spanning the threshold that distinguishes positive from negative selection. We show that, at the selection threshold, a small increase in ligand affinity for the T-cell antigen receptor leads to a marked change in the activation and subcellular localization of Ras and mitogen-activated protein kinase (MAPK) signalling intermediates and the induction of negative selection. The ability to compartmentalize signalling molecules differentially in the cell endows the thymocyte with the ability to convert a small change in analogue input (affinity) into a digital output (positive versus negative selection) and provides the basis for establishing central tolerance.

The perception and response of T cells to a changing environment are based on the law of initial value.

αß T cells are critical components of the adaptive immune system and are capable of inducing sterilizing immunity after pathogen infection and eliminating transformed tumor cells. The development and function of T cells are controlled through the T cell antigen receptor, which recognizes peptides displayed on major histocompatibility complex (MHC) molecules. Here, we review how T cells generate the ability to recognize self-peptide-bound MHC molecules and use signals derived from these interactions to instruct cellular development, activation thresholds, and functional specialization in the steady state and during immune responses. We argue that the basic tenants of T cell development and function follow Weber-Fetcher's law of just noticeable differences and Wilder's law of initial value. Together, these laws argue that the ability of a system to respond and the quality of that response are scalable to the basal state of that system. Manifestation of these laws in T cells generates clone-specific activation thresholds that are based on perceivable differences between homeostasis and pathogen encounter (self versus nonself discrimination), as well as poised states for subsequent differentiation into specific effector cell lineages.

Tracking cryptic SARS-CoV-2 lineages detected in NYC wastewater.

Tracking SARS-CoV-2 genetic diversity is strongly indicated because diversifying selection may lead to the emergence of novel variants resistant to naturally acquired or vaccine-induced immunity. To monitor New York City (NYC) for the presence of novel variants, we deep sequence most of the receptor binding domain coding sequence of the S protein of SARS-CoV-2 isolated from the New York City wastewater. Here we report detecting increasing frequencies of novel cryptic SARS-CoV-2 lineages not recognized in GISAID's EpiCoV database. These lineages contain mutations that had been rarely observed in clinical samples, including Q493K, Q498Y, E484A, and T572N and share many mutations with the Omicron variant of concern. Some of these mutations expand the tropism of SARS-CoV-2 pseudoviruses by allowing infection of cells expressing the human, mouse, or rat ACE2 receptor. Finally, pseudoviruses containing the spike amino acid sequence of these lineages were resistant to different classes of receptor binding domain neutralizing monoclonal antibodies. We offer several hypotheses for the anomalous presence of these lineages, including the possibility that these lineages are derived from unsampled human COVID-19 infections or that they indicate the presence of a non-human animal reservoir.

The persistence of T cell memory.

T cell memory is a crucial feature of the adaptive immune system in the defense against pathogens. During the last years, numerous studies have focused their efforts on uncovering the signals, inflammatory cues, and extracellular factors that support memory differentiation. This research is beginning to decipher the complex gene network that controls memory programming. However, how the different signals, that a T cell receives during the process of differentiation, interplay to trigger memory programming is still poorly defined. In this review, we focus on the most recent advances in the field and discuss how T cell receptor signaling and inflammation control CD8 memory differentiation.