Adjuvant Delivery Method and Nanoparticle Charge Influence Peptide Amphiphile Micelle Vaccine Bioactivity.

Vaccines are an indispensable public health measure that have enabled the eradication, near elimination, and prevention of a variety of pathogens. As research continues and our understanding of immunization strategies develops, subunit vaccines have emerged as exciting alternatives to existing whole vaccine approaches. Unfortunately, subunit vaccines often possess weak antigenicity, requiring delivery devices and adjuvant supplementation to improve their utility. Peptide amphiphile micelles have recently been shown to function as both delivery devices and self-adjuvanting systems that can be readily associated with molecular adjuvants to further improve vaccine-mediated host immunity. While promising, many "design rules" associated with the plethora of underlying adjustable parameters in the generation of a peptide amphiphile micelle vaccine have yet to be uncovered. This work explores the impact micellar adjuvant complexation method and incorporated antigen type have on their ability to activate dendritic cells and induce antigen specific responses. Interestingly, electrostatic complexation of CpG to micelles resulted in improved in vitro dendritic cell activation over hydrophobic association and antigen|adjuvant co-localization influenced cell-mediated, but not antibody-mediated immune responses. These exciting results complement those previously published to build the framework of a micelle vaccine toolbox that can be leveraged for future disease-specific formulations.

Lower female survival from an opportunistic infection reveals progesterone-driven sex bias in trained immunity.

Immune responses differ between females and males, although such sex-based variance is incompletely understood. Observing that bacteremia of the opportunistic pathogen Burkholderia gladioli caused many more deaths of female than male mice bearing genetic deficiencies in adaptive immunity, we determined that this was associated with sex bias in the innate immune memory response called trained immunity. Female attenuation of trained immunity varies with estrous cycle stage and correlates with serum progesterone, a hormone that decreases glycolytic capacity and recall cytokine secretion induced by antigen non-specific stimuli. Progesterone receptor antagonism rescues female trained immune responses and survival from controlled B. gladioli infection to magnitudes similar to those of males. These data demonstrate progesterone-dependent sex bias in trained immunity where attenuation of female responses is associated with survival outcomes from opportunistic infection.

NOTCH signaling in COVID-19: a central hub controlling genes, proteins, and cells that mediate SARS-CoV-2 entry, the inflammatory response, and lung regeneration.

In the lungs of infected individuals, the downstream molecular signaling pathways induced by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are incompletely understood. Here, we describe and examine predictions of a model in which NOTCH may represent a central signaling axis in lung infection in Coronavirus Disease 2019 (COVID-19). A pathway involving NOTCH signaling, furin, ADAM17, and ACE2 may be capable of increasing SARS-CoV-2 viral entry and infection. NOTCH signaling can also upregulate IL-6 and pro-inflammatory mediators induced to hyperactivation in COVID-19. Furthermore, if NOTCH signaling fails to turn down properly and stays elevated, airway regeneration during lung healing can be inhibited-a process that may be at play in COVID-19. With specific NOTCH inhibitor drugs in development and clinical trials for other diseases being conducted, the roles of NOTCH in all of these processes central to both infection and healing merit contemplation if such drugs might be applied to COVID-19 patients.

Early expression of mature αß TCR in CD4-CD8- T cell progenitors enables MHC to drive development of T-ALL bearing NOTCH mutations.

During normal T cell development in mouse and human, a low-frequency population of immature CD4-CD8- double-negative (DN) thymocytes expresses early, mature αß T cell antigen receptor (TCR). We report that these early αß TCR+ DN (EADN) cells are DN3b-DN4 stage and require CD3δ but not major histocompatibility complex (MHC) for their generation/detection. When MHC - is present, however, EADN cells can respond to it, displaying a degree of coreceptor-independent MHC reactivity not typical of mature, conventional αß T cells. We found these data to be connected with observations that EADN cells were susceptible to T cell acute lymphoblastic leukemia (T-ALL) transformation in both humans and mice. Using the OT-1 TCR transgenic system to model EADN-stage αß TCR expression, we found that EADN leukemogenesis required MHC to induce development of T-ALL bearing NOTCH1 mutations. This leukemia-driving MHC requirement could be lost, however, upon passaging the tumors in vivo, even when matching MHC was continuously present in recipient animals and on the tumor cells themselves. These data demonstrate that MHC:TCR signaling can be required to initiate a cancer phenotype from an understudied developmental state that appears to be represented in the mouse and human disease spectrum.

Conservation and Enhanced Binding of SARS-CoV-2 Omicron Spike Protein to Coreceptor Neuropilin-1 Predicted by Docking Analysis.

The Omicron variant of SARS-CoV-2 bears peptide sequence alterations that correlate with a higher infectivity than was observed in the original SARS-CoV-2 isolated from Wuhan, China. We analyzed the CendR motif of spike protein and performed in silico molecular docking with neuropilin-1 (Nrp1), a receptor-ligand interaction known to support infection by the original variant. Our analysis predicts conserved and slightly increased energetic favorability of binding for Omicron CendR:Nrp1. We propose that the viral spike:Nrp1 coreceptor pathway may contribute to the infectivity of the Omicron variant of SARS-CoV-2.

Vasoactive Intestinal Peptide Amphiphile Micelle Chemical Structure and Hydrophobic Domain Influence Immunomodulatory Potentiation.

Vasoactive intestinal peptide (VIP) is a neuropeptide capable of downregulating innate immune responses in antigen presenting cells (APCs) by suppressing their pro-inflammatory cytokine secretion and cell surface marker expression. Though VIP's bioactivity could possibly be leveraged as a treatment for transplant tolerance, drug delivery innovation is required to overcome its intrinsically limited cellular delivery capacity. One option is to employ peptide amphiphiles (PAs) which are lipidated peptides capable of self-assembling into micelles in water that can enhance cellular association. With this approach in mind, a series of triblock VIP amphiphiles (VIPAs) has been synthesized to explore the influence of block arrangement and hydrophobicity on micelle biocompatibility and bioactivity. VIPA formulation has been found to influence the shape, size, and surface charge of VIPA micelles (VIPAMs) as well as their cytotoxicity and immunomodulatory effects. Specifically, the enclosed work provides strong evidence that cylindrical VIPAMs with aspect ratios of 1.5-150 and moderate positive surface charge are able to potentiate the bioactivity of VIP limiting TNF-α secretion and MHC II and CD86 surface expression on APCs. With these criteria, we have identified PalmK-(EK)4-VIP as our lead formulation, which showed comparable or enhanced anti-inflammatory effects relative to the unmodified VIP at all dosages evaluated. Additionally, the relationships between peptide block location and lipid block size provide further information on the chemical structure-function relationships of PA micelles for the delivery of VIP as well as potentially for other peptides more broadly.

Non-Randomized Trial of Dornase Alfa for Acute Respiratory Distress Syndrome Secondary to Covid-19.

Background: The most severe cases of Coronavirus-Disease-2019 (COVID-19) develop into Acute Respiratory Distress Syndrome (ARDS). It has been proposed that oxygenation may be inhibited by extracellular deoxyribonucleic acid (DNA) in the form of neutrophil extracellular traps (NETs). Dornase alfa (Pulmozyme, Genentech) is recombinant human deoxyribonuclease I that acts as a mucolytic by cleaving and degrading extracellular DNA. We performed a pilot study to evaluate the effects of dornase alfa in patients with ARDS secondary to COVID-19. Methods: We performed a pilot, non-randomized, case-controlled clinical trial of inhaled dornase for patients who developed ARDS secondary to COVID-19 pneumonia. Results: Improvement in arterial oxygen saturation to inhaled fraction of oxygen ratio (PaO2/FiO2) was noted in the treatment group compared to control at day 2 (95% CI, 2.96 to 95.66, P-value = 0.038), as well as in static lung compliance at days 3 through 5 (95% CI, 4.8 to 19.1 mL/cmH2O, 2.7 to 16.5 mL/cmH2O, and 5.3 to 19.2 mL/cmH2O, respectively). These effects were not sustained at 14 days. A reduction in bronchoalveolar lavage fluid (BALF) myeloperoxidase-DNA (DNA : MPO) complexes (95% CI, -14.7 to -1.32, P-value = 0.01) was observed after therapy with dornase alfa. Conclusion: Treatment with dornase alfa was associated with improved oxygenation and decreased DNA : MPO complexes in BALF. The positive effects, however, were limited to the time of drug delivery. These data suggest that degradation of extracellular DNA associated with NETs or other structures by inhaled dornase alfa can be beneficial. We propose a more extensive clinical trial is warranted. Clinical Trial Registration: ClinicalTrials.gov, Identifier: NCT04402970.

Transcriptional Regulation of the Human IL5RA Gene through Alternative Promoter Usage during Eosinophil Development.

Regulation of the IL-5 receptor alpha (IL5RA) gene is complicated, with two known promoters (P1 and P2) driving transcription, and two known isoforms (transmembrane and soluble) dichotomously affecting the signaling potential of the protein products. Here, we sought to determine the patterns of P1 and P2 promoter usage and transcription factor occupancy during primary human eosinophil development from CD34+ hematopoietic stem cell progenitors. We found that during eosinophilopoiesis, both promoters were active but subject to distinct temporal regulation, coincident with combinatorial interactions of transcription factors, including GATA-1, PU.1, and C/EBP family members. P1 displayed a relatively constant level of activity throughout eosinophil development, while P2 activity peaked early and waned thereafter. The soluble IL-5Rα mRNA peaked early and showed the greatest magnitude fold-induction, while the signaling-competent transmembrane isoform peaked moderately. Two human eosinophilic cell lines whose relative use of P1 and P2 were similar to eosinophils differentiated in culture were used to functionally test putative transcription factor binding sites. Transcription factor occupancy was then validated in primary cultures by ChIP. We conclude that IL-5-dependent generation of eosinophils from CD34+ precursors involves complex and dynamic activity including both promoters, several interacting transcription factors, and both signaling and antagonistic protein products.

Chronic respiratory disease disparity between American Indian/Alaska Native and white populations, 2011-2018.

BACKGROUND: American Indian/Alaska Native (AI/AN) populations have been disproportionately affected by chronic respiratory diseases for reasons incompletely understood. Past research into disease disparity using population-based surveys mostly focused on state-specific factors. The present study investigates the independent contributions of AI/AN racial status and other socioeconomic/demographic variables to chronic respiratory disease disparity in an 11-state region with historically high AI/AN representation. Using data from the Behavioral Risk Factor Surveillance System (BRFSS) spanning years 2011-2018, this work provides an updated assessment of disease disparity and potential determinants of respiratory health in AI/AN populations. METHODS: This cross-sectional study used data from the BRFSS survey, 2011-2018. The study population included AI/AN and non-Hispanic white individuals resident in 11 states with increased proportion of AI/AN individuals. The yearly number of respondents averaged 75,029 (62878-87,350) which included approximately 5% AI/AN respondents (4.5-6.3%). We compared the yearly adjusted prevalence for chronic respiratory disease, where disease status was defined by self-reported history of having asthma and/or chronic obstructive pulmonary disease (COPD). Multivariable logistic regression was performed to determine if being AI/AN was independently associated with chronic respiratory disease. Covariates included demographic (age, sex), socioeconomic (marital status, education level, annual household income), and behavioral (smoking, weight morbidity) variables. RESULTS: The AI/AN population consistently displayed higher adjusted prevalence of chronic respiratory disease compared to the non-Hispanic white population. However, the AI/AN race/ethnicity characteristic was not independently associated with chronic respiratory disease (OR, 0.93; 95% CI, 0.79-1.10 in 2017). In contrast, indicators of low socioeconomic status such as annual household income of <$10,000 (OR, 2.02; 95% CI, 1.64-2.49 in 2017) and having less than high school education (OR, 1.37; 95% CI, 1.16-1.63 in 2017) were positively associated with disease. These trends persisted for all years analyzed. CONCLUSIONS: This study highlighted that AI/AN socioeconomic burdens are key determinants of chronic respiratory disease, in addition to well-established risk factors such as smoking and weight morbidity. Disease disparity experienced by the AI/AN population is therefore likely a symptom of disproportionate socioeconomic challenges they face. Further promotion of public health and social service efforts may be able to improve AI/AN health and decrease this disease disparity.

Public and private human T-cell clones respond differentially to HCMV antigen when boosted by CD3 copotentiation.

Human cytomegalovirus (HCMV) induces long-lasting T-cell immune responses that control but do not clear infection. Typical responses involve private T-cell clones, expressing T-cell antigen receptors (TCRs) unique to a person, and public T-cell clones with identical TCRs active in different people. Here, we report the development of a pretherapeutic immunostimulation modality against HCMV for human T cells, CD3 copotentiation, and the clonal analysis of its effects in recall assays at single-cell resolution. CD3 copotentiation of human T cells required identification of an intrinsically inert anti-CD3 Fab fragment that conditionally augmented signaling only when TCR was coengaged with antigen. When applied in recall assays, CD3 copotentiation enhanced the expansion of both public and private T-cell clones responding to autologous HLA-A2(+) antigen-presenting cells and immunodominant NLVPMVATV (NLV) peptide from HCMV pp65 protein. Interestingly, public vs private TCR expression was associated with distinct clonal expansion signatures in response to recall stimulus. This implied that besides possible differences in their generation and selection in an immune response, public and private T cells may respond differently to pharmacoimmunomodulation. Furthermore, a third clonal expansion profile was observed upon CD3 copotentiation of T-cell clones from HLA-A2(-) donors and 1 HLA-A2(+) presumed-uninfected donor, where NLV was of low intrinsic potency. We conclude that human T-cell copotentiation can increase the expansion of different classes of T-cell clones responding to recall antigens of different strengths, and this may be exploitable for therapeutic development against chronic, persistent infections such as HCMV.

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation.

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Eosinophils downregulate lung alloimmunity by decreasing TCR signal transduction.

Despite the accepted notion that granulocytes play a universally destructive role in organ and tissue grafts, it has been recently described that eosinophils can facilitate immunosuppression-mediated acceptance of murine lung allografts. The mechanism of eosinophil-mediated tolerance, or their role in regulating alloimmune responses in the absence of immunosuppression, remains unknown. Using lung transplants in a fully MHC-mismatched BALB/c (H2d) to C57BL/6 (H2b) strain combination, we demonstrate that eosinophils downregulate T cell-mediated immune responses and play a tolerogenic role even in the absence of immunosuppression. We further show that such downregulation depends on PD-L1/PD-1-mediated synapse formation between eosinophils and T cells. We also demonstrate that eosinophils suppress T lymphocyte responses through the inhibition of T cell receptor/CD3 (TCR/CD3) subunit association and signal transduction in an inducible NOS-dependent manner. Increasing local eosinophil concentration, through administration of intratracheal eotaxin and IL-5, can ameliorate alloimmune responses in the lung allograft. Thus, our data indicate that eosinophil mobilization may be utilized as a novel means of lung allograft-specific immunosuppression.

The early proximal αß TCR signalosome specifies thymic selection outcome through a quantitative protein interaction network.

During αß T cell development, T cell antigen receptor (TCR) engagement transduces biochemical signals through a protein-protein interaction (PPI) network that dictates dichotomous cell fate decisions. It remains unclear how signal specificity is communicated, instructing either positive selection to advance cell differentiation or death by negative selection. Early signal discrimination might occur by PPI signatures differing qualitatively (customized, unique PPI combinations for each signal), quantitatively (graded amounts of a single PPI series), or kinetically (speed of PPI pathway progression). Using a novel PPI network analysis, we found that early TCR-proximal signals distinguishing positive from negative selection appeared to be primarily quantitative in nature. Furthermore, the signal intensity of this PPI network was used to find an antigen dose that caused a classic negative selection ligand to induce positive selection of conventional αß T cells, suggesting that the quantity of TCR triggering was sufficient to program selection outcome. Because previous work had suggested that positive selection might involve a qualitatively unique signal through CD3δ, we reexamined the block in positive selection observed in CD3δ0 mice. We found that CD3δ0 thymocytes were inhibited but capable of signaling positive selection, generating low numbers of MHC-dependent αß T cells that expressed diverse TCR repertoires and participated in immune responses against infection. We conclude that the major role for CD3δ in positive selection is to quantitatively boost the signal for maximal generation of αß T cells. Together, these data indicate that a quantitative network signaling mechanism through the early proximal TCR signalosome determines thymic selection outcome.

Breaking tolerance with engineered class I antigen-presenting molecules.

Successful efforts to activate T cells capable of recognizing weak cancer-associated self-antigens have employed altered peptide antigens to activate T cell responses capable of cross-reacting on native tumor-associated self. A limitation of this approach is the requirement for detailed knowledge about the altered self-peptide ligands used in these vaccines. In the current study we considered allorecognition as an approach for activating CTL capable of recognizing weak or self-antigens in the context of self-MHC. Nonself antigen-presenting molecules typically contain polymorphisms that influence interactions with the bound peptide and TCR interface. Recognition of these nonself structures results in peptide-dependent alloimmunity. Alloreactive T cells target their inducing alloantigens as well as third-party alloantigens but generally fail to target self-antigens. Certain residues located on the alpha-1/2 domains of class I antigen-presenting molecules primarily interface with TCR. These residues are more conserved within and across species than are residues that determine peptide antigen binding properties. Class I variants designed with amino acid substitutions at key positions within the conserved helical structures are shown to provide strong activating signals to alloreactive CD8 T cells while avoiding changes in naturally bound peptide ligands. Importantly, CTL activated in this manner can break self-tolerance by reacting to self-peptides presented by native MHC. The ability to activate self-tolerant T cells capable of cross-reacting on self-peptide-MHC in vivo represents an approach for inducing autoimmunity, with possible application in cancer vaccines.

Clustering the autisms using glutamate synapse protein interaction networks from cortical and hippocampal tissue of seven mouse models.

Background: Autism spectrum disorders (ASDs) are a heterogeneous group of behaviorally defined disorders and are associated with hundreds of rare genetic mutations and several environmental risk factors. Mouse models of specific risk factors have been successful in identifying molecular mechanisms associated with a given factor. However, comparisons among different models to elucidate underlying common pathways or to define clusters of biologically relevant disease subtypes have been complicated by different methodological approaches or different brain regions examined by the labs that developed each model. Here, we use a novel proteomic technique, quantitative multiplex co-immunoprecipitation or QMI, to make a series of identical measurements of a synaptic protein interaction network in seven different animal models. We aim to identify molecular disruptions that are common to multiple models. Methods: QMI was performed on 92 hippocampal and cortical samples taken from seven mouse models of ASD: Shank3B, Shank3Δex4-9, Ube3a2xTG, TSC2, FMR1, and CNTNAP2 mutants, as well as E12.5 VPA (maternal valproic acid injection on day 12.5 post-conception). The QMI panel targeted a network of 16 interacting, ASD-linked, synaptic proteins, probing 240 potential co-associations. A custom non-parametric statistical test was used to call significant differences between ASD models and littermate controls, and Hierarchical Clustering by Principal Components was used to cluster the models using mean log2 fold change values. Results: Each model displayed a unique set of disrupted interactions, but some interactions were disrupted in multiple models. These tended to be interactions that are known to change with synaptic activity. Clustering revealed potential relationships among models and suggested deficits in AKT signaling in Ube3a2xTG mice, which were confirmed by phospho-western blots. Conclusions: These data highlight the great heterogeneity among models, but suggest that high-dimensional measures of a synaptic protein network may allow differentiation of subtypes of ASD with shared molecular pathology.

iNKT cells ameliorate human autoimmunity: Lessons from alopecia areata.

Alopecia areata (AA) is understood to be a CD8+/NKG2D+ T cell-dependent autoimmune disease. Here, we demonstrate that human AA pathogenesis of is also affected by iNKT10 cells, an unconventional T cell subtype whose number is significantly increased in AA compared to healthy human skin. AA lesions can be rapidly induced in healthy human scalp skin xenotransplants on Beige-SCID mice by intradermal injections of autologous healthy-donor PBMCs pre-activated with IL-2. We show that in this in vivo model, the development of AA lesions is prevented by recognized the iNKT cell activator, α-galactosylceramide (α-GalCer), which stimulates iNKT cells to expand and produce IL-10. Moreover, in pre-established humanized mouse AA lesions, hair regrowth is promoted by α-GalCer treatment through a process requiring both effector-memory iNKT cells, which can interact directly with CD8+/NKG2D+ T cells, and IL-10. This provides the first in vivo evidence in a humanized model of autoimmune disease that iNKT10 cells are key disease-protective lymphocytes. Since these regulatory NKT cells can both prevent the development of AA lesions and promote hair re-growth in established AA lesions, targeting iNKT10 cells may have preventive and therapeutic potential also in other autoimmune disorders related to AA.

Science Signaling Podcast for 2 August 2016: Patient-specific protein complexes.

This Podcast features an interview with Adam Schrum and Steven Neier, authors of a Research Article that appears in the 2 August 2016 issue of Science Signaling, about a method for identifying protein-protein interactions in patient tissue samples. The authors used this method to compare signaling complexes downstream of the T cell receptor in T cells from healthy skin with those in T cells from the skin of patients with the autoimmune disease alopecia areata. The study revealed differences in the relative abundance of some protein complexes between T cells from the control and patient groups. This technique could be adapted for use as a diagnostic tool to stratify patients by molecular phenotype and predict the therapeutic strategy that is likely to work best for each patient.Listen to Podcast.

Multiplex matrix network analysis of protein complexes in the human TCR signalosome.

Multiprotein complexes transduce cellular signals through extensive interaction networks, but the ability to analyze these networks in cells from small clinical biopsies is limited. To address this, we applied an adaptable multiplex matrix system to physiologically relevant signaling protein complexes isolated from a cell line or from human patient samples. Focusing on the proximal T cell receptor (TCR) signalosome, we assessed 210 pairs of PiSCES (proteins in shared complexes detected by exposed surface epitopes). Upon stimulation of Jurkat cells with superantigen-loaded antigen-presenting cells, this system produced high-dimensional data that enabled visualization of network activity. A comprehensive analysis platform generated PiSCES biosignatures by applying unsupervised hierarchical clustering, principal component analysis, an adaptive nonparametric with empirical cutoff analysis, and weighted correlation network analysis. We generated PiSCES biosignatures from 4-mm skin punch biopsies from control patients or patients with the autoimmune skin disease alopecia areata. This analysis distinguished disease patients from the controls, detected enhanced basal TCR signaling in the autoimmune patients, and identified a potential signaling network signature that may be indicative of disease. Thus, generation of PiSCES biosignatures represents an approach that can provide information about the activity of protein signaling networks in samples including low-abundance primary cells from clinical biopsies.

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.