Cancer immunotherapy has revolutionized the treatment landscape in medical oncology, but its efficacy has been variable across patients. Biomarkers to predict such differential response to immunotherapy include cytotoxic T lymphocyte infiltration, tumor mutational burden, and microsatellite instability. A growing number of studies also suggest that baseline tumor burden, or tumor size, predicts response to immunotherapy. In this review, we discuss the changes in immune profile and therapeutic responses that occur with increasing tumor size. We also overview therapeutic approaches to reduce tumor burden and favorably modulate the immune microenvironment of larger tumors. Nearly 50 years ago, Whitney and colleagues reported that mice bearing large carcinogen-induced sarcomas displayed defective spontaneous immune responses . These iThe complex immunobiology of the TME is regulated by a number of factors, including tumor cell-intrinsic determinants of immune cell infiltration , the tisvia immune cell subsets recruited or induced within the TME. Pro-tumorigenic myeloid derived suppressor cells (MDSCs) mediate T cell suppression through a variety of mechanisms including depletion of arginine, oxidative stress of target cells, and release of the dicarbonyl radical methylglyoxal , a pleiotropic cytokine shown to exert anti-tumor effects in early-stage cancer but tumor-promoting effects in late-stage cancer , is amplHowever, not all TMEs follow the same positive correlation between suppressor cell infiltration and increased tumor size. In patients with colorectal cancer, CXCL9 – an IFN-γ inducible chemokine that recruits CD8 T cells – is not differentially expressed according to high or low tumor burden . In a movia negative immune checkpoints including programmed death-1 (PD-1) or cytotoxic T lymphocyte-associated protein 4 (CTLA-4). Ligation of immune checkpoints on T cells drives exhaustion and dysfunction, preventing autoimmunity and immunopathology in the context of extenuating immune activation, but usurped by the tumor to disable anti-tumor immunity. Immune checkpoint ligands such as programmed death ligand 1 (PD-L1) are abundant in the TME of many tumor types, independently of tumor mutation burden  to disrupt the ligand/receptor pairing of these immune checkpoint molecules enables tumor-associated T cells to overcome immunosuppression and effectively perform anti-tumor functions . ICB hasDespite the immense positive impact of ICB in the clinical setting, many patients do not respond, and tumor burden is one metric that negatively correlates with ICB efficacy. Preclinical data reveals that PD-1 blockade is more effective in mice bearing smaller lung squamous cell tumors . Similarvia ICB, direct co-stimulation of T cells using mAbs targeting molecules such as OX40 and 4-1BB has also been explored. OX40 is a member of the TNF receptor superfamily expressed by activated T cells, and agonistic OX40 mAbs have direct stimulatory activities on effector T cells with the benefit of inhibiting Treg function in the tumor site, showing early promise in clinical trials (2) require the addition of transforming growth factor-β (TGF-β) receptor antagonists to further reduce immune suppression  that works prophylactically or as a very early therapeutic intervention for small CT26 colon carcinomas was insufficient to eradicate large (>70mm3) established tumors (in situ priming against tumor antigens via cytokine release), results in better outcomes if tumor burden is low at the start of therapy  to improve T cell priming, with the underlying hypothesis that insufficient T cell priming is one reason for the failure of ICB or co-stimulation mAbs to “rescue” T cell responses in the TME  has shown promise in metastatic melanoma  is an appealing therapeutic option that bypasses the requirement for melanoma , 72. Howmelanoma . In largmor size . Similarmor size . Recent mor size .In addition to TILs, genetically modified T cells can also be used for ACT, including CAR T cells, where success has been made in many hematological malignancies, but solid tumors remain a more difficult problem . HematolGiven that large tumors are more resistant to immunotherapy and harbor greater populations of suppressive cells, targeting tumor burden-associated alterations in the tumor microenvironment may exert therapeutic effects. Here, we briefly cover two major categories of approaches that may improve immune responses against tumors: directly reducing tumor size and modulating the tumor immune microenvironment.Tumor resection is the oldest method of treating cancer, and one of the original methods of restoring defective tumor surveillance in mouse models . The useCytotoxic chemotherapy is another viable strategy to reduce tumor size that has been employed in the clinic for decades. Moreover, some chemotherapy regimens drive immunogenic cell death of tumor cells, leading to release of tumor antigens that can be presented by APCs to prime cytotoxic T cells . In thisHowever, given the cytotoxic nature of chemotherapy on any proliferating cell including T cells , 87, theRadiotherapy delivers high-energy rays directly to tumor sites as a form of curative control locally at the tumor, and the resultant abscopal effect can be beneficial for immune responses. The administration of radiation treatment completely ablates the tumor and surrounding stroma, effectively debulking the tumors. The immune impact is therefore complex—immune suppression may be alleviated by the eradication of the local tumor, while increased tumor cell death enhances systemic anti-tumor T cell response by generating novel tumor antigens presented on major histocompatibility complex (MHC)-I . In prec3) and small (50 mm3) mouse CT26 colon carcinomas as compared to anti-PD-L1, which was only effective against small tumors  play a similarly suppressive role in the TME and can also be targeted using depletion strategies. Colony-stimulating factor-1 (CSF-1), a key regulator of macrophage differentiation, can be blocked  killing . Skewing killing . Paradox killing , 117. Ho killing  and bols+ CD4 T cells. While effector CD4 T cell depletion does not negatively impact all immunotherapies  are canonical pattern recognition receptors, and ligation by toll-like agonists stimulate the innate immune sensing pathways. In the context of the cancer-immunity cycle, providing TLR agonists licenses APCs to enhance antigen presentation and production of inflammatory cytokines . TLR agoinations . Furtherinations .Despite these positive changes in the TME after TLR agonist administration, there are some tumor cells that express—and are activated—by TLR ligation , 131, whA second class of innate immune sensor stimulation bypasses the binding of TLRs and instead directly stimulates the cyclic GMP-AMP synthase (cGAS) - stimulator of IFN genes (STING) pathway, which is triggered in the presence of cytosolic DNA . Activatvia type I IFNs. In contrast, CD40 stimulation bypasses the use of innate immune sensors and Type I IFN signaling, thus presenting an alternate bridge between innate and adaptive immunity , originally designed to specifically kill tumor cells, have more recently been utilized as potent activators of local and systemic immune responses. OVs drive tumor cell death, resulting in the release of inflammatory signals (cell stress danger-associated molecular patterns or DAMPS) and the liberation of tumor-associated antigens to promote anti-tumor immunity. Most recently, clinical trials in patients with brain tumors have shown promising results using OVs , such asvia tumor cell lysis (resulting in the liberation of tumor antigens and DAMPs) and the recruitment of DCs . Intereoduction  and is boduction  and via e trials . Recentle trials . It has e trials . HoweverTargeting proteins downstream of RAS in the MAPK pathway has been more successful. BRAFV600E is one of the most common mutations, and a class of RAF monomer inhibitors specifically target this mutation to block mutant BRAF signaling . Vemurafin vitro and in lymph nodes  inhibition controls tumor progression, but has been shown to negatively impact T cell priming and proliferation ph nodes . However, which regulate chromatin remodeling, are aberrantly expressed in many cancer types. HDACi results in cytostatic or cytotoxic effects on tumor cells, but this approach has been more effective in hematological malignancies than in solid tumors when used as a monotherapy or combined with other drugs including chemotherapy, reviewed by Suraweera and colleagues . Despitein vitro . Importain vitro . The impin vitro . These sin vitro studies revealed a cytotoxic impact after blocking autophagy in cancer cells and the first clinical trial was run in glioblastoma where patient survival was significantly extended when CQ was combined with radiation and chemotherapy (Autophagy is a mechanism by which cancer cells (and other cell types) degrade intracellular organelles as a source of nutrients under both basal and stress-induced conditions . Two FDAotherapy , and CQ otherapy . In a mootherapy . Combiniotherapy , revealiotherapy , the benotherapy . CD8 T cotherapy , while Dotherapy , highligClinical and pre-clinical data indicate that tumor burden negatively correlates with response to a range of immunotherapies that include ICB, adoptive T cell therapy, and activation of antigen-presenting cells. Mechanistically, large tumors exert greater local and systemic changes to the immune system, and harbor more immunosuppressive cells and molecules that dampen antitumor activity. Many of the alterations locally and systemically reflect a more immunosuppressive tumor microenvironment; however, integrating this within the context of tumor burden may help stratify patients for optimally designed combination therapy approaches. Targeting features of the “large” TME can skew the immune microenvironment more toward phenotype of a “small” TME, rendering the tumor more sensitive to immunotherapeutic interventions as highlighted in SK and KB wrote the manuscript; all authors edited the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Roy and Diana Vagelos Scholars Program in the Molecular Life Sciences at the University of Pennsylvania (SK) and Parker Institute for Cancer Immunotherapy (KB).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


How T lymphocytes tune their responses to different strengths ofstimulation is a fundamental question in immunology. Recent work using newoptogenetic, single-cell genomic and live-imaging approaches has revealed thatstimulation strength controls the rate of individual cell responses within apopulation. Moreover, these responses have been found to use shared molecularprograms, regardless of stimulation strength. However, additional data indicatethat stimulation duration or cytokine feedback can impact later gene expressionphenotypes of activated cells. In-depth molecular studies have suggestedmechanisms by which stimulation strength might modulate the probability of Tcell activation. This emerging model allows activating T cells to achieve a widerange of population responses through probabilistic control within individualcells. TCRligation rapidly recruits signaling molecules to trigger a broad and interconnectednetwork of signaling events including protein phosphorylation and calciumfluxes  including cytokine production and, for cytotoxic Tlymphocytes (CTLs), secretion of cytolytic proteins at theimmunological synapse. Each of theseactivation events occurs within individual cells, with the sum ofindividual responses creating a population response. This review highlights newtechnologies and the insights they have revealed, suggesting how TCR-pMHCinteractions in single cells can generate finely-tuned activation responses within apopulation, and focusing on the early hours after TCR ligation in naïve andeffector T cells.How T cells meet the challenge of integrating signals from a seeminglyinfinite array of pathogens with only a limited set of intracellular machinery haslong puzzled immunologists. T cell receptors (TCRs) on the cell surface need tosense both the quantity and quality of peptide-MHC  that exhibits the desired binding behavior, makingquestions about specific lengths of pMHC engagement or patterns of binding andre-binding events difficult to answer. To circumvent this issue, several groups haverecently developed optogenetic receptor-ligand systems in which bindingkinetics are controlled by light patterns  and protein kinase D2 (PKD2). Other markers of activation showa graded response such that increasing stimulation strength shifts the markerintensity within each individual cell. IRF4 expression is the best characterized ofthese “analog” responses, with extensive studies inmurine CD8+ T cells. Recently, a hybrid digital/analogmodel has been used to describe certain activation markers that exhibit both“on/off” behavior and graded modulation of intensity within the“on” population (e.g. expression of CD69 in CD4+ Tcells and MYC inCD8+ T cells). For these markers, both thepercentage of positive cells and the intensity of the positive population areinfluenced by stimulation strength. The existence of such hybrid behaviors suggeststhat the digital/analog dichotomy may be overly simplistic. This is particularlyrelevant for gene expression changes, which can accumulate over the course of activesignaling, as describedin detail below.One of the original single-cell methods, activationrate (events per unit time) of a response. We argue that thesedistinctions are crucial when testing the impact of stimulation strength onactivation phenotypes, as they can lead to different interpretations ofhow the underlying intracellular machinery reads TCR signals.While flow cytometry has been instrumental in revealing these activationbehaviors, its early use had two major drawbacks. First, early flow cytometrymethods produced uni- or oligo-dimensional measurements, leaving the relationshipsbetween activation events within individual cells unclear.  and mass cytometry , as wellin vitro naïveCD8+ T cell activation, using the OTI TCR-transgenic mouse system, in which all T cells are specificfor an ovalbumin peptide and for which APLs of varied affinities have beenwell-characterized.Using pseudotime analyses to compare the activation progress of cellsstimulated with different APLs, this study demonstrated that transcriptionalresponses to strong stimulation were rapid and synchronized while responses to weakstimulation were more temporally heterogeneous and on average delayed. However, the transcriptionalactivation trajectory was largely shared, regardless of stimulation strength,suggesting that this process is utilized by all activating cells. These resultsindicate that stimulation strength can impact the rate with which cells initiatetranscriptional activation.In naïve T cells, one of the primary outcomes of TCR stimulation isthe induction of gene expression. A recent study scRNA-seq study examined thetranscriptional changes downstream of . This approach revealed a set of signaling eventsthat were shared among cells regardless of stimulation strength, but which were, onaverage, delayed with weaker stimuli. These results echo the transcriptionalfindings that stimulation strength can control the rate with which cells initiate ashared activation program. Due to the rapidity and transience of proximal signalingevents, this latter study focused on TCR-distal signaling nodes, honing in on thecoordination of ERK, S6 and STAT5 phosphorylation, and future work examining simultaneous activationof TCR-proximal signaling mediators will be important to understand the initiationof this shared downstream signaling program.A subsequent mass cytometry study looked upstream of transcriptionalactivation and asked how signaling events marked by protein phosphorylation anddegradation across multiple T cell signaling pathways were influenced by ligandaffinity in the same OTI T cell activation system. Likewise, results from thesehigh-dimensional single-cell studies are reminiscent of earlier work that useddivision-tracking dye to monitor proliferation of APL-stimulated OTI T cells andfound that the rate of proliferation entry, but not the speed of ongoingproliferation, was dependent on stimulation strength. Together these studies demonstrate that undercontrolled in vitro settings, stimulation strength can regulate therate of activation in naïve CD8+ T cells. Comparison within vivo studies will be important to understand how such amechanism plays out in a complex physiological environment.Similar conservation of T cell activation processes was observed in flowcytometry experiments examining markers of metabolic shift and cell cycle entry inOTI TCR-transgenic cells stimulated with APLs of varied affinities. One study used confocal liveimaging of in vitro-activated OTI TCR-transgenic CTLs to monitorthe impact of ligand affinity on the dynamics of the CTL-target cell interaction andthe intracellular movement of the centrosome and granules. Data showed that TCR stimulation strength wasassociated with the proportion of cells exhibiting long dwell times,sustained calcium fluxes, docked centrosomes, and polarized granules. However,within cells that achieved long dwell times and organelle polarization, theorganization and speed of the response was independent of stimulation strength,suggesting a conserved activation program. These results suggest that, as innaïve T cells, stimulation strength controls the rate of effector CTLactivation. Such a rate-based model . Moreover, extensive work using APLs to stimulatenaïve CD8+ T cells in multiple murine TCR-transgenic systems hasshown that starting from approximately one day after activation, T cells expressIRF4 in a graded manner reflecting stimulation strength. As IRF4can enhance effector differentiation, this suggeststhat subtle tuning of gene expression in the early days of naïve T cellactivation might alter differentiation outcomes, as has been observed in invivo models for both CD4+ and CD8+ Tcells.Similarly, experiments stimulating murine CD4+ T cells with varyingantigen doses found that after 24 hours, stimulation strength correlated with theexpression of IL-12Rß2, which facilitates Th1 polarization in response toIL-12 signaling. These resultsagain suggest a means by which stimulation strength can impact differentiation fate.Together, these data indicate that although shared activation programs may exist,the strength of T cell stimulation can further tune resulting activated T cellphenotypes.Additional evidence of stimulus-dependent tuning beyond a shared activationprogram comes from studies of TCR-induced gene expression changes. In the scRNA-seqstudy described above, after accounting for each cell’s activation status, asmall number of genes remained differentially expressed at the mRNA level betweencells stimulated by strong and weaker ligands. Using an optogenetic chimeric antigenreceptor (optoCAR), in which light induced the dissociation of theintracellular signaling moiety from the receptor-ligand complex and its subsequentinactivation, the authors quantified the persistence of TCR-induced signalsincluding calcium flux, ERK and FOS phosphorylation, and gene transcription, in thehuman Jurkat T cell line. Results showed that upon proximal signaling disruption,downstream activation events rapidly dissipated, but sustained signaling led to theaccumulation of gene expression outputs in the hours following activation. Whilestudies in primary cells using TCRs will be required to determine thegeneralizability of these findings, they suggest that stimulation strength couldimpact mRNA and protein expression phenotypes by altering the effective duration ofstimulation that cells experience.This raises the important question, if the rate-based model for activationis accurate, how are responses tuned beyond a core activation program according tostimulus? One potential explanation is that cells continue to receive stimulationbeyond an initial activation event. An elegant optogenetic study tested the impactof sustained signaling on T cell activation responsesin vitro stimulation of murineTCR-transgenic CD4+ T cells with mathematical modeling found that theavailability of the effector-promoting cytokine IL-2 is carefully regulated according to antigen dosethrough multiple feedback loops. Suchdifferences in the cytokine milieu might mediate strength-dependent cellularresponses, particularly at later time points when stimulation-induced cytokinescould feed back on the activating cells. Further exploration of tuning behaviors andthe conditions in which they are observed will thus be important to betterunderstand the full impacts of stimulation strength on T cell activation.An alternative though not mutually exclusive explanation for stimulationstrength-dependent response tuning is that the T cell microenvironment, and thus theadditional signals the cell receives, changes with stimulation strength. Forexample, previous work combining + T cells specific for a peptide from moth cytochrome C and pMHCon supported planar lipid bilayers additionally functionalized withICAM-1. They found awide distribution of receptor-ligand dwell times, the mean of which corresponded toTCR-pMHC affinity. Moreover, measurements of nuclear translocation of thetranscription factor NFAT (as an activation marker) revealed that successfulactivation was associated with either a single long dwell time, or sequential,short, spatially-correlated binding events. In this way, all activated cellsreceived the same total input, regardless of ligand affinity. The model suggested bythis study is that activation events occur in a probabilistic manner, taking placewhen sufficiently long real or effective dwell times are stochastically achieved.This interpretation provides an intriguing mechanism that might explain how ligandaffinity as well as concentration can alter the rate of cellular activation.Observations that stimulation strength can control the rate with which Tcells initiate a core activation program suggest a switch-like mechanism at somestage of the TCR-induced signaling pathway where the decision to signal furtherdownstream is made. Recent live-imaging work has shed light on the TCR ligationproperties that modulate this switch. Specifically, one study used TIRF microscopyto image individual TCR-pMHC interactions between murine TCR-transgenicCD4kinetic proofreading, which posits thatsignaling steps introduce a delay between ligand binding and subsequent activationcascades, such that weak interactions often dissociate before responses aretriggered. Two recent optogenetic studiesexplicitly tested the concept of kinetic proofreading in T cells using light toalter the binding half-lives of synthetic ligand-receptor pairs in an otherwiseuniform environment. One study used a LOVTRAPsystem in which a CAR expressed in Jurkat cells was bound in alight-controlled manner to LOV2 presented on a supported lipid bilayer. The second study used aPhyB/PIF system in which Jurkat cells expressed a construct of PIF6fused to a TCRß chain that underwent light-controlled binding to PhyBtetramers. Both studiesfound that longer binding half-lives resulted in greater activation, even whencontrolling for receptor occupancy, consistent withthe kinetic proofreading model. However, it must be noted that there are manydifferences between these synthetic receptor systems and native T cell-APCinteractions, which include coreceptors and adhesion molecules among other factors.Thus, continued testing of the model in native systems is merited.Theoretically, converting TCR-pMHC binding dwell times into a highlydiscriminatory activation switch requires a thresholding mechanism. One of the mostpopular models for this is in vitroexperiments and mathematical modeling to calculate the number of steps required forkinetic proofreading. Theauthors varied ligand dose and affinity while stimulating primary humanCD8+ T cells expressing an exogenous TCR, and then fitted a model ofa kinetic proofreading mechanism. This yielded an estimate that the delay frominitial TCR binding to activation takes 2.8 seconds and 2.67 biochemical steps . These results suggest that if a molecular switch exists, it occursearly in the signaling pathway. Testing whether data from other T cell stimulationsystems yield the same parameter estimates will be important to gauge thegeneralizability of this conclusion.The stage in the signaling network at which kinetic proofreading might beachieved also remains unclear. A recent study combined . Experimentsvarying the number of ITAMs on synthetic receptors expressed in Jurkat cells foundthat increasing the number of ITAMs increased the proportion of cells exhibitingactivation phenotypes, including NFAT reporter expression and ERK phosphorylation,and the synchronicity of activation. These results appear similar to those seen with increasingligand affinity, suggesting that the signal strength conferred by ligand affinitymight impact the efficiency of TCR ITAM phosphorylation. Following ITAMphosphorylation, ZAP70 is recruited and activated. Experiments using the LOVTRAP optoCAR describedabove and measuring ZAP70 recruitment and diacylglycerol (DAG) accumulation inresponse to varied dwell times and receptor occupancy levels revealed no evidence ofkinetic proofreading at the level of ZAP70 recruitment, but offered strong evidenceof this mechanism further downstream at the level of DAG accumulation. These data suggest that theputative molecular switch is between these two activation events in this optoCARsystem and provide a hypothesis for testing in intact T cells.Following TCR ligation, phosphorylation of immunoreceptor tyrosine-basedactivation motifs (ITAMs) in the intracellular portions of CD3 subunits initiatessignaling cascadesLAT signalosome assembles, recruitingand activating multiple signaling intermediates including Phospholipase C gamma 1(PLCγl), which cleaves phosphatidylinositol 4,5-bisphosphate P2) togenerate DAG and inositol 1,4,5-trisphosphate (IP3). Intriguing recent evidencesuggests that phosphorylation of LAT Y132 in humans (LAT Y136 in mice) might beresponsible for initiating the T cell activation program (reviewed in ref). Phosphorylation of most LAT tyrosines ispromoted by neighboring acidic residues, but Y132 is an exception and is phosphorylated at a slowerrate. The substitution of an acidic residue next toY132 was sufficient to enhance its phosphorylation rate, and increase thephosphorylation, recruitment, and activation of PLCγ1 . With this modification, both Jurkat cells and primarymurine CD8+ T cells that had no or poor responses to weak ligandstimulation in the absence of the acidic residue now activated. These results suggest that thisLAT phosphorylation event might act as a molecular switch controlling T cellactivation. A subsequent mathematical modeling study confirmed the importance ofthis phosphorylation step in ligand discrimination and hypothesizednumerically-supported mechanisms by which it might either form a kineticproofreading step itself, or sustain proofreading from an earlier step. This study also highlighted thenecessity of spatial colocalization of proximal signaling mediators to achievekinetic proofreading. Together these results raise the possibility that kineticproofreading might occur at or upstream of the slow phosphorylation of LAT Y132,focusing the field for future investigations.Downstream of ZAP70, the , an event that was further exacerbated by the addition ofPLCγ1. Second, aconfocal live imaging study in murine in vitro-differentiated CTLsfound that the catalytic activity of PLCγ1 at the CTL immune synapse drives apositive feedback mechanism.This study tracked lipid modifications at the immunological synapse over time andexogenously expressed a modified PIP5K with constitutive synapse localization.Their results showed that the PLCγ1-induced reduction in negatively chargedPIP2 causes a loss of electrostatically bound PIP5K, preventing regeneration ofthe negative charge and depleting the actin mesh across the synapse, allowinggranule secretion to occur. Subsequent work in the same system showed that reducingstimulation strength reduced the area of synapse depleted of negative charge andactin, as well as the proportion of cells capable of achieving thisdepletion, suggestingthat the efficiency of this process depends on stimulation strength. Finally, thoughsimultaneous imaging of centrosome movement and calcium flux, this latter studyrevealed a calcium flux threshold associated with centrosome docking, which, together with previousreports implicating both DAG and the calcium flux in centrosomepolarization, suggests a tipping point at orupstream of PLCγ1. Thus, although it remains an open question, there isincreasing evidence to suggest that slow modification of LAT and recruitment ofPLCγ1 might constitute a gateway to downstream activation programs,converting stimulation strength into a probability of activation at the single-celllevel.Further evidence that PLCγ1 recruitment to the LAT signalosome maymark a turning point in T cell activation comes from both its interaction with LATand its ability to cleave PIP2 into DAG and IP3, which drives the calcium flux.First, experiments monitoring the condensation of LAT on supported lipid bilayers inthe presence of GRB2 and SOS showed that the formation of LAT aggregates can act asa rate-limiting step in the activation of RASprobabilistic model for initiating amolecular program has been proposed in the context of in vivo Tcell differentiation, where individual cells exhibited extensive heterogeneity intheir progress along a shared differentiation trajectory, but the combinedpopulation response was highly robust. Moreover, the use of fixed molecular programs has precedent inother biological systems, for example development, where integrated signalingnetworks can control activation of a consistent set of differentiationpathways. As T cells continue to integrate signals fromtheir TCR and other environment-sensing receptors, it is highly likely that furthertuning of responses takes place beyond a core activation program – themechanisms of which are not fully understood. It also remains unclear how tunableactivation responses are at the individual cell level. As described in theOutstanding Questions, further dissection through use of these andother emerging technologies will continue to shed light on the regulatory logicgoverning T cell responses, which may benefit our understanding of diseases drivenby inappropriate T cell activation as well as inform the rational design of Tcell-targeting therapeutics or vaccines.Recent advances in single-cell genomic and imaging technologies, combinedwith greater control over T cell stimulation, have enabled researchers to revisit Tcell activation questions from a newly dynamic and granular perspective. Thisvantagepoint has revealed that stimulation strength can impact the rate with whichcells utilize a common set of activation programs. These observations can reconcileresults from previous studies using bulk, static, or uni-dimensional measurementsthat found differences in the speed, magnitude, or proportion of T cell responses.Such a mechanism is intellectually appealing as it enables a wide range of T cellresponses at the population level without requiring infinitely diverse responsesfrom each individual cell. A


Long non-coding RNAs (lncRNAs) are key regulators of diverse cellular processes. Although a number of studies have reported the identification of bovine lncRNAs across many tissues, very little is known about the identity and characteristics of lncRNAs in bovine oocytes.A bovine oocyte cDNA library was constructed and sequenced using the Illumina HiSeq 2000 sequencing system. The oocyte transcriptome was constructed using the ab initio assembly software Scripture and Cufflinks. The assembled transcripts were categorized to identify the novel intergenic transcripts, and the coding potential of these novel transcripts was assessed using CPAT and PhyloCSF. The resulting candidate long intergenic non-coding RNAs (lincRNAs) transcripts were further evaluated to determine if any of them contain any known protein coding domains in the Pfam database. RT-PCR was used to analyze the expression of oocyte-expressed lincRNAs in various bovine tissues.A total of 85 million raw reads were generated from sequencing of the bovine oocyte library. Transcriptome reconstruction resulted in the assembly of a total of 42,396 transcripts from 37,678 genomic loci. Analysis of the assembled transcripts using the step-wide pipeline resulted in the identification of 1535 oocyte lincRNAs corresponding to 1183 putative non-coding genes. A comparison of the oocyte lincRNAs with the lncRNAs reported in other bovine tissues indicated that 970 of the 1535 oocyte lincRNAs appear to be unique to bovine oocytes. RT-PCR analysis of 5 selected lincRNAs showed either specific or predominant expression of 4 lincRNAs in the fetal ovary. Functional prediction of the oocyte-expressed lincRNAs suggested their involvement in oogenesis through regulating their neighboring protein-coding genes.This study provides a starting point for future research aimed at understanding the roles of lncRNAs in controlling oocyte development and early embryogenesis in cattle. Over the past decade, genome-wide transcriptional studies have discovered that the vast majority of the mammalian genome (up to 80%) is transcribed, while only 2–3% of the mammalian genome is transcribed into protein-coding RNAs (mRNAs) , 2. The Bos taurus) is one of the most commonly raised livestock for meat, milk and other dairy products. As lncRNAs play a key role in the regulation of gene expression, it is important to identify and characterize bovine lncRNAs. To date, a number of studies have reported the identification of bovine lncRNAs across many tissues [Cattle  and metaphase II (MII) stage oocytes were obtained using procedures as described previously . Total RProtein-coding genes were downloaded from UCSC genome browser  and EnseAfter trimming adaptor sequences and filtering rRNAs and ambiguous and low-quality bases, a total of 78 million pair-end reads were obtained. Spliced read aligner TopHat2 was used to align all clean reads to the bovine genome (UMD3.1) using the default parameters. Aligned reads from TopHat2 were assembled into transcriptome by Scripture  and CuffIdentification of each transcript as either coding or noncoding was performed using a step-wise pipeline to filter out the transcripts that had a high chance of being protein coding. First, all transcripts that had exon overlapping a transcript from any of the following sets were eliminated: coding genes annotated in UCSC, RefSeq and Ensembl, and microRNAs, tRNAs, snoRNAs and rRNAs annotated in Ensembl. Second, coding potential of each candidate transcript was assessed using PhyloCSF  and CPATTo evaluate tissue specificity of a transcript, an entropy-based metric that relies on Jensen-Shannon (JS) divergence was used to calculate specificity scores (0 to 1). A perfect tissue-specific pattern is scored as JS = 1, which means a transcript is expressed only in one tissue .18 primer and Superscript III reverse transcriptase . The cDNA was used for PCR amplification using lincRNA-specific primers  using TopHat2 . 78.4%  of the oocyte lincRNAs identified in this study overlap with previously reported bovine lncRNAs . The FPKM (fragments per kilobase of transcript per million mapped reads) value of each transcript in each of the 9 tissues was calculated by Cufflinks . As shown in Fig. cis and regulate the expression of a neighboring protein coding gene [cis regulation hypothesis is that the expression between lincRNA and its neighboring gene would be correlated across all samples used in the present study. To predict the potential functions of the oocyte lincRNAs, a total of 1239 lincRNAs with a JS score larger than 0.5 were selected. We then screened ~ 50 kb genomic region as neighboring chromosome region [cis.Recent studies suggest that some lincRNAs may act in ing gene , 40. Thee region  flankinge region . A totalIn this study, we presented the comprehensive annotation of bovine oocyte lincRNAs using RNA-Seq data from bovine oocytes. In order to assess tissue specificity of newly identified lincRNAs in bovine oocytes, we collected RNA-Seq data sets from multiple bovine tissues from NCBI RSA database. The tissue-specificity score was calculated based on the FPKM for each transcript and demonstrated that bovine oocyte lincRNAs are expressed in a much more tissue-specific manner. It would be interesting to find out if oocyte lncRNAs in other species, such as humans, show similar expression pattern. Similar studies could be conducted in humans using RNA sequencing data from human oocytes. Such data have become available recently .cis to regulate the gene expression in their chromosomal neighborhood, we were able to predict the putative functions for 75 oocyte lincRNAs. Most importantly, we found that cytoskeleton organization, regulation of microtubule-based process, zinc ion transport and mitochondrial respiratory chain complex assembly were over-represented for neighboring genes of oocyte-specific lincRNAs. Early embryonic development in many organisms relies on the subcellular organization of the oocyte and requires the coordination of a variety of cellular events. Cytoskeleton was believed to mediate many of these processes. More importantly, microtubules, a component of the cytoskeleton, are the major constituents of spindles that are used to pull apart eukaryotic chromosomes during mitosis and meiosis. Moreover, Stephenson et al. [Based on the hypothesis that lincRNAs might act in n et al.  reportedn et al. . Insteadn et al. .Reversible phosphorylation is important in regulating oocyte meiosis. The inhibition of phosphatase-1 (PP1) and PP2A was found to stimulate oocyte germinal vesicle breakdown . PhosphoOocyte-specific genes are known to play important roles in folliculogenesis, fertilization, and early embryonic development . This stIn the present study, we performed ab initio assembly of more than 80 million RNA-Seq reads from bovine GV and MII stage oocytes and identified 1535 transcribed lincRNAs from 1183 loci. In addition, we calculated the tissue specificity score for each oocyte lincRNA and demonstrated that the majority of oocyte lincRNAs (80%) are tissue-specific. Finally, we proposed functions of oocyte specific lincRNAs, suggesting their involvement in oogenesis through regulating their neighboring protein-coding genes. This study provides a foundation for future investigations on the roles of oocyte-expressed lncRNAs in controlling oocyte development and early embryogenesis in cattle.Additional file 1: Table S1. List of lincRNAs identified in bovine oocytes.Additional file 2: Table S2. Comparison of bovine oocyte lincRNAs with previously identified lncRNAs in NONCODEv4 database.Additional file 3: Table S3. Expression of lincRNAs in bovine oocytes relative to 9 other tissues.


Allogeneic hematopoietic stem cell transplantation (aHSCT) is a curative therapy for a range of hematologic illnesses including aplastic anemia, sickle cell disease, immunodeficiency, and high-risk leukemia, but the efficacy of aHSCT is often undermined by graft-versus-host disease (GVHD), where T cells from the donor attack and destroy recipient tissues. Given the strong interconnection between T cell metabolism and cellular function, determining the metabolic pathways utilized by alloreactive T cells is fundamental to deepening our understanding of GVHD biology, including its initiation, propagation, and potential mitigation. This review summarizes the metabolic pathways available to alloreactive T cells and highlights key metabolic proteins and pathways linking T cell metabolism to effector function. Our current knowledge of alloreactive T cell metabolism is then explored, showing support for glycolysis, fat oxidation, and glutamine metabolism but also offering a potential explanation for how these presumably contradictory metabolic findings might be reconciled. Examples of additional ways in which metabolism impacts aHSCT are addressed, including the influence of butyrate metabolism on GVHD resolution. Finally, the caveats and challenges of assigning causality using our current metabolic toolbox is discussed, as well as likely future directions in immunometabolism, both to highlight the strengths of the current evidence as well as recognize some of its limitations. Allogeneic hematopoietic stem cell transplantation (aHSCT) is a potentially curative therapy for a wide range of hematologic maladies ranging from genetic diseases to aggressive leukemias and lymphomas –3. An unin vivo T cell biology. This review will highlight our current understanding of alloreactive T cell metabolism in light of the major metabolic pathways, present evidence for involvement of various pathways at distinct stages of the process, define key metabolic regulators that influence substrate choice, and integrate multiple lines of evidence into a cohesive overarching hypothesis. We close by highlighting examples of additional ways in which metabolism can influence GVHD and discuss challenges to the interpretation of metabolic data.It is broadly accepted that metabolism and immune cell function are linked, with immunologic differentiation influencing immune cell metabolism and metabolic pathways impacting immune responses , 12. It Cellular metabolism is a complex interplay between multiple different enzymes, substrates, intermediates, and end products. Classically, glycolysis, and oxidative phosphorylation (OXPHOS) have been studied as the primary pathways that supply cellular energy. Glycolysis consists of a series of enzymatic steps that convert glucose into pyruvate. Depending on the intrinsic and extrinsic needs of the cell, pyruvate can then either be converted into lactate and excreted from the cell or channeled into acetyl-coA and further oxidized via OXPHOS. While lactate fermentation classically occurs in oxygen poor environments, T cells can perform glycolysis and lactate fermentation in oxygen replete environments, referred to as aerobic glycolysis. Although glycolysis might not be the best choice based solely on energy production, glycolytic intermediates can also act as substrates for anabolic pathways including amino acid synthesis, nucleotide synthesis, and the pentose phosphate pathway (PPP) , all pro2  by donating electrons to Complex I and II  cycle uses the end products of glycolysis, fatty acid oxidation, and glutamine metabolism to generate the reducing intermediates, NADH, and FADH2 . NADH anI and II , a proceI and II .regs) rely on oxidation of fatty acids and glucose to maintain their energetic balance , a glucose analog, identifies increased tracer uptake in the gastrointestinal tract during GVHD in both mice and humans (eff) function, while increased glucose trafficking may be of potential diagnostic value. The caveat is that glycolytic metabolism may also prove essential to the function of non-GVHD T cells.Since aerobic glycolysis is critical for physiologic T cell activation , 27, 28,ow alone . In addid humans . Thus, iin vitro skewing conditions but still readily differentiate into FoxP3+ Treg cells , a serine/threonine kinase belonging to the phosphoinositol 3-kinase (PI3K) family. mTOR is the catalytic subunit for either of two distinct protein complexes; mTORC1 (with scaffolding protein Raptor) and mTORC2 (with scaffolding protein RICTOR) . mTORC1 eg cells . Furthereg cells , while meg cells , 48. Theeg cells , and agamTOR has also been implicated in promoting the pathogenicity of alloreactive T cells. In pre-clinical models, mTOR activity increases in alloreactive T cells and both the pharmacologic inhibition of mTOR, and its genetic deletion, inhibit glycolysis without impacting OXPHOS , 49. TheIn addition to glucose metabolism, there is ample evidence that alloreactive T cells rely on the energy and by-products of OXPHOS. This necessity of OXPHOS, in addition to an increase in lactate fermentation, is likely necessary due to the greater energetic demands experienced by T cells undergoing constant exposure to high levels of antigen in a near continuous manner. In allogeneic cells, oxygen consumption increased markedly compared to either naïve T cells or T cells recovered from syngeneic recipients . MitochoAlthough the mechanisms that influence OXPHOS are complex, the cellular energy sensor AMP-activated protein kinase (AMPK) has been implicated as a driver of oxidative metabolism and could play a role. AMPK is heterotrimeric protein complex consisting of a serine/threonine kinase α subunit, a stabilizing β subunit, and a regulatory γ subunit. The γ subunit detects low intracellular energy levels by sensing the AMP/ATP ratio (increased during low energy states) and responds by facilitating activation of the AMPKα kinase domain . AMPK acListeria monocytogenes infection model, T cell-specific deletion of AMPKα1 impaired memory CD8 cell generation compared to wildtype T cells without impacting primary immune responses  but did not impact T cell development or differentiation . Later sesponses . More re in vivo  and has  in vivo . AMPK hactivator . Metformrthritis , 66. Howreg cells recovered from metformin-treated recipients  Treg increase in number but are minimally suppressive, allowing effector responses to proceed unabated. As the infection subsides, inflammatory signals decrease and FoxP3 levels stabilize, leading to the adoption of OXPHOS, which decreases Treg proliferation but increases suppressive function, limiting the effector response and restoring a state of tolerance , which controls mitochondrial DNA copy number and is integral to ETC activity , mTOR is also required for proper Treg function and development. Compared to conventional T cells, Treg have higher mTORC1 activity  is a class III histone deacetylase whose expression influences multiple metabolic pathways. Donor T cells lacking Sirt1 show increased FoxP3 stability in inducible Treg (iTreg) with a subsequent decreased conversion to pathogenic IFN-γ producing cells and a loss of follicular helper T cell development  effect. It is expected that anything that interrupts T cell alloreactivity, or impairs allogeneic T cell number, might disrupt the therapeutic efficacy of allogeneic transplantation. And yet metabolic manipulation does not appear to be universally detrimental to anti-cancer responses. Donor T cells that lack AMPK induce less severe GVHD but continue to demonstrate preserved or even enhanced cytotoxic potential post-transplant . Similarreg induction in the gastrointestinal tract is influenced by production of short chain fatty acids (SCFAs), commonly produced by commensal bacteria, and primarily in the forms of butyrate and propionate. SCFAs induce Foxp3 expression , essentially improving the host response to injury. In a follow-up study, GPR43 expression was found to be necessary to realize the GVHD-protective effects of butyrate and because of this necessity GVHD severity increased in mice lacking GPR43  populations  promoter. In both cases, genetic deletion occurs during T cell development, leaving a long time for T cells to utilize alternative compensatory pathways, akin to taking the back-roads into work when the highway is unavailable. This point is important to keep in mind, as experiments using genetic knockouts might easily yield results confounded by the adoption of compensatory pathways.mem and Treg cells developed in vitro and in vivo in the absence of CPT1a (presumably lacking FAO). While these findings appear to contradict a necessity for FAO in Treg and Tmem cells  , 123, whex vivo prior to transplantation into recipients. Leveraging this advantage, metabolic inhibitors or activators could be applied exclusively to donor cells, sparing tissues from systemic administration of the compound. Furthermore, it may be possible to inhibit a first pathway through ex vivo manipulation , followed by subsequent inhibition of a compensatory pathway in vivo through systemic administration of a second inhibitor . Cells lesioned in the first pathway would be expected to be more sensitive to secondary inhibition, while T cells arising de novo (and not having experienced the primary inhibition) would be spared. Ultimately, precise metabolic modulation and the potential for simultaneous inhibition of multiple metabolic pathways, will enhance the efficacy of metabolism-based therapies.Finally, careful study of T cell metabolism as well as secondary/tertiary compensatory pathways will improve the timing and specificity of our inhibition. In this regard, aHSCT offers the distinct advantage of having a period in which donor cells are manipulated The breadth of research highlighted thus far lays a strong foundation for an increased understanding of T cell metabolism during aHSCT. However, outstanding questions remain regarding the heterogeneity of T cells recovered post-transplant and whether murine findings are translatable to humans. Like many complex disease processes, T cells collected and analyzed post-aHSCT represent a heterogenous population of cells including those driving the alloreactive response and bystanders simply responding to the inflammatory milieu. Studying metabolism in whole T cells during GVHD thus glosses over differences between individual T cell subsets and complicates the understanding of metabolism in the most highly alloreactive T cells. One approach to clarify this issue has been to use GVHD models in which donor T cells respond to a specific antigen. For example, transgenic CD8 T cells that recognize the SIINFEKL peptide of ovalbumin  can be injected into CAG-OVA recipients which express ovalbumin as a self-antigen. Using this model, T cells transplanted into CAG-OVA recipients expressed increased levels of ROS and PD-1 compared to OT-1 T cells responding to immunization with CAG-OVA expressing dendritic cells . In a diAnother approach is to use single-cell metabolomics to determine the individual metabolic profiles of donor T cells isolated from allogeneic recipients, a breakthrough that has the potential to revolutionize the study of immunometabolism during GVHD. While there are many groups working to develop single-cell metabolic technologies, the approach remains relatively new and faces numerous challenges, including the technical hurdle of how to collect cells without altering their metabolite abundance –129. To in vivo. Increased use of xGVHD models will undoubtedly increase the likelihood that experimental findings will apply to human patients. However, xGVHD models themselves are imperfect and do not improve entirely upon deficiencies of classical animal models. Thus, patient samples may prove to be the best way to validate laboratory findings and ensure that observations are applicable to real world scenarios. In recent years, the repertoire of available technologies for in vivo human work has expanded, making the in vivo study of T cell metabolism during GVHD more feasible than ever. For example, recent publications have used administration of non-radioactive isotope tracers to study tissue metabolism in human patients in real time  improve upon existing animal models by injecting human peripheral blood mononuclear cells (hPBMCs) into immunodeficient mice , allowineal time , 133. Itreg exhibit a distinct metabolic profile linking oxidative metabolism to T cell suppressive function, a phenomenon that appears to hold during allogeneic transplantation, yet remains the focus of intense investigation. In the end, studying T cell metabolism in the context of GVHD will help to deepen our understanding of in vivo T cell biology and identify novel therapies for the treatment of T cell-mediated pathologies.This review has investigated recent developments in our understanding of alloreactive T cell metabolism. While evidence suggests that many metabolic pathways are active in alloreactive T cells , includiRB reviewed the literature, drafted the manuscript, and reviewed the final product. CB reviewed and interpreted the primary literature, edited the manuscript draft, and critically revised the final manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cholangiocarcinoma (CCA) represents a disease entity that comprises a heterogeneous group of biliary malignant neoplasms, with variable clinical presentation and severity. It may be classified according to its anatomical location and distinguished in intrahepatic (iCCA), perihilar (pCCA), or distal (dCCA), each subtype implying distinct epidemiology, biology, prognosis, and strategy for clinical management. Its incidence has increased globally over the past few decades, and its mortality rate remains high due to both its biological aggressiveness and resistance to medical therapy. Surgery is the only potentially curative treatment and is the standard approach for resectable CCA; however, more than half of the patients have locally advanced or metastatic disease at presentation. For patients with unresectable CCA, the available systemic therapies are of limited effectiveness. However, the advances of the comprehension of the complex molecular landscape of CCA and its tumor microenvironment could provide new keys to better understand the pathogenesis, the mechanisms of resistance and ultimately to identify promising new therapeutic targets. Recently, clinical trials targeting isocitrate dehydrogenase (IDH)-1 mutations and fibroblast growth factor receptor (FGFR)-2 fusions, as well as immunotherapy showed promising results. All these new and emerging therapeutic options are herein discussed. Cholangiocarcinoma (CCA) is a group of epithelial cell malignancies arising from cholangiocellular epithelium, most of which are adenocarcinoma . It is aCCA is subclassified as intrahepatic cholangiocarcinoma (iCCA), originating from the biliary tree within the liver, and extrahepatic cholangiocarcinoma (eCCA), outside the liver parenchyma; the latter is further subdivided into perihilar cholangiocarcinoma (pCCA or Klatskin tumor) and distal cholangiocarcinoma (dCCA), with a frequency of 10–20% iCCA, 50% pCCA and 30–40% dCCA  after first-line chemotherapy. The overall response rate was 14.8%, almost all with FGFR2 fusions, and median progression-free survival was 5.8 months, and interestingly disease control rate was 75.4%; however, the durability of response was limited  . IncludeThere are other ongoing trials exploring the role of other checkpoint inhibitors as durvalumab in solid tumors including CCA .In this phase 1 study preliminary results , the disCombinational strategies between different ICIs or a combination of an ICI with a chemo-therapy backbone seem to be a promising strategy that is also under exploration.CCAs are known to be surrounded by a reactive tumor stroma, containing cancer-associated fibroblasts, endothelial cells, and immune cells, including tumor-associated macrophages (TAMs). These stromal elements produce soluble factors that play a role in modulating anticancer immune responses as reported by a small retrospective study involving 39 patients with CCA, in which high numbers of alternatively activated, ‘M2-like’ TAMs were reported to be associated with worse disease-free survival. Based on these observations, the combination of immunotherapy and microenvironmental targeting  and pegylated IFNα-2b (Peg-IFNα-2b), fibroblast growth factor receptors (FGFR1–3) and heat-shock protein 90 (HSP90)) might be an effective treatment option and some ongoing trials are exploring this hypothesis.Promising results have been reported through the administration of immunotherapies in malignancies commonly associated with viral infections  and the However, further studies are needed to draw more robust conclusions, particularly to define which subgroup of CCA might most benefit from immunotherapy and to identify specific predictors of tumor response.The final results of the clinical trials studying ICIs in CCA setting are still ongoing. Even if the preliminary results have been quite modest when evaluating the nude overall response rate, these studies are still limited by the single-arm design, the small sample sizes, the unavailability of MSI/MMR status, and heterogeneous or unreported characteristics of the primary tumor, i.e., intrahepatic vs. extrahepatic or gallbladder.On the other hand, the heterogeneity of CCA may represent a limit for evaluating the response rate, as the different tumors show differential responses to the same therapies. Therefore, targeting the immune system may be highly variable according to the different genetic profiles of the tumor and the microenvironment and sub-analyses of large cohorts of patients treated with ICIs are needed to identify the factors associated with response.Despite a complex tumor and immune microenvironment with features suggesting the potential for antitumor immune responses, ICI monotherapy has shown limited efficacy in CCA to date, though the safety profile has been reassuring. Moreover, more complex approaches with combinations of different agents may lead to more clinical impact.Advanced CCA remains a difficult-to-treat disease. Future studies will continue focusing on therapies targeting specific genetic aberrations  . We stilThe value of molecular markers of the tumor in identifying these genetic aberrations also requires further study.The next generation of clinical trials—studying chemotherapy, target-therapy, immunotherapy or their combination aimed at getting better antitumor response are still ongoing .Gemcitabine plus cisplatin remains the standard first-line systemic therapy for advanced CCA and offers a median survival of approximately one year. No standard regimens beyond the first line and no targeted or immunotherapy agents are approved yet in this disease. The development of molecular targeted therapy in this heterogeneous and relatively rare malignancy continues to be a challenging area. The rapidly growing precision medicine efforts have uncovered the underlying mutational landscape of this difficult to treat disease and paved the way for molecularly oriented clinical trials.


Otitis media (OM) is a common reason for children to be prescribed antibiotics and undergo surgery but a thorough understanding of disease mechanisms is lacking. We evaluate the evidence of a dysregulated immune response in the pathogenesis of OM.A comprehensive systematic review of the literature using search terms [otitis media OR glue ear OR AOM OR OME] OR [middle ear AND (infection OR inflammation)] which were run through Medline and Embase via Ovid, including both human and animal studies. In total, 82 955 studies underwent automated filtering followed by manual screening. One hundred studies were included in the review.Most studies were based on in vitro or animal work. Abnormalities in pathogen detection pathways, such as Toll-like receptors, have confirmed roles in OM. The aetiology of OM, its chronic subgroups  and recurrent acute OM is complex; however, inflammatory signalling mechanisms are frequently implicated. Host epithelium likely plays a crucial role, but the characterisation of human middle ear tissue lags behind that of other anatomical subsites.Translational research for OM presently falls far behind its clinical importance. This has likely hindered the development of new diagnostic and treatment modalities. Further work is urgently required; particularly to disentangle the respective immune pathologies in the clinically observed phenotypes and thereby work towards more personalised treatments. Otitis media (OM) is an umbrella term for inflammation of the middle ear and is a leading cause of medical consultations, antibiotic prescriptions and surgery in children Ref. . WorldwiBox 1.Glossary of termsThe following terms are based upon definitions provided in Ref.  and Ref.Most patients exhibit complete resolution of infective symptoms of acute OM within a few days. However, a subgroup subsequently suffers from ongoing pathology, including (1) recurrent acute OM, in which there are regular episodes of acute infection causing pain but with resolution of symptoms in between. (2) A persistent fluid effusion in the middle ear ; this impairs the conduction of sound through the middle ear which leads to hearing loss and is associated with a negative impact on language acquisition, behaviour and progress at school Ref. . (3) ChrDespite the considerable burden of disease, it presently remains unclear why the majority of children make a spontaneous recovery from limited episodes of acute OM but a subset, who are frequently otherwise apparently immunocompetent, develop recurrent acute or chronic disease.In health, the human immune system regulates the response to infectious stimuli by providing robust action against non-self-antigens whilst exerting tolerance to the host's own cells. However, at many anatomical sites, dysfunction of this process drives many inflammatory disease processes Ref. . Here weA comprehensive systematic review of the literature using search terms [otitis media OR glue ear OR AOM OR OME] OR [middle ear AND (infection OR inflammation)] were run through Medline and Embase via Ovid. Both human and animal studies were included. In total, 82 955 studies underwent automated filtering for the English language only articles, full-text availability only, dated 1990–2021. Following de-duplication, 2557 abstracts were manually screened. One hundred studies were included in the review, including those identified in bibliographies of returned studies for a comprehensive literature review. A summary PRISMA diagram is available in Supplementary materials.An initial episode of acute OM may be viral or bacterial in nature, and the former can predispose to the latter Ref. . ClinicaThe origin of these organisms is likely from the nasopharynx via the Eustachian tube , as demoIn order to generate an immune response, the host must first detect the presenting pathogen. This is mediated by pattern-recognition receptors (PRRs) such as membrane-bound Toll-like receptors (TLRs) or C-type lectins which are expressed on diverse host cell types including epithelial cells, as well as classical immune sentinels such as macrophages and dendritic cells. They detect invariant microbial motifs or pathogen-associated molecular patterns (PAMPs) in the extracellular space and trigger signal transduction via adaptor proteins such as myeloid differentiation primary response protein 88 (MyD88), leading to a pro-inflammatory response Ref. .Tlr9 knockout models show a prolonged inflammatory response in the middle ear and delayed bacterial clearance , have been shown to be essential for resolution of OM in mice  and Tir-domain-containing adaptor inducing interferon ice Refs , 18. SimAOM Ref. . The mulMoraxella catarrhalis has been shown to deliver outer membrane vesicles in response to TLR2 binding, and surface proteins – such as ubiquitous surface protein A1 (UspA1) – modulate the inflammatory response of epithelial cells. It therefore seems that otopathogens can interact with specific TLRs to subdue the host response to infection once they have infiltrated the tissue, making them challenging to eradicate  or interferon regulatory factor 3 (IRF3) which act as transcription factors to initiate a pro-inflammatory and type 1 interferon response which are essential for effective bacterial and viral clearance , which is secreted by tissue resident macrophages or indeed infected tissue itself. Consequently, IL-8 in middle ear effusion is significantly more elevated in culture-positive acute OM than culture-negative OM Ref. , and is Epithelial cells have important interactions with immune cells and consequently modulate airway inflammation. For example, healthy airway epithelium is a potent inhibitor of dendritic cell-induced T-cell activation Ref. . FurtherInsight from other upper airway mucosal sites may also be informative in understanding epithelial inflammation in OM. In chronic rhinosinusitis, for example, there is now emerging evidence of distinct endotypes which are each mediated by particular innate lymphoid cell (ILC1/2/3) and T helper cell (Th1/Th2/Th17) subgroups. It logically follows that treatments for these subgroups may differ – particularly with the advent of targeted monoclonal antibody or ‘biologic’ therapies Ref. . Analogoγ than those from children without OM , which are responsible for chemoattraction of inflammatory cells, are significantly more elevated in middle ear effusion in persistent OM but whether this is cause or effect remains unclear Ref. . Other wAlternative hypotheses can be derived from the observation that OME is more prevalent in children with concomitant allergic rhinitis than those without Refs , 78, sugAs with pathogen detection, the mechanisms governing mucosal inflammation in COM are less well understood than in AOM and OME. It is clear that the host middle ear epithelium is crucial for tissue homeostasis, the regulation of inflammation and antimicrobial defence, and is therefore likely of importance Ref. . It is, Tlr2, 4 and 5, but the upregulation of TNF-ɑ, IL-1β, IFN-γ and IL-6 compared with healthy ears. Together these indicate a pro-inflammatory milieu which, over time, leads to histological and functional changes which diminish the host's capacity to respond to infection in those with COM  pathway with agonist Ciglitazone which dampens the inflammatory response in the human middle ear epithelial cell (HMEECs) line in vitro  has shown promise in reducing cellular infiltrate, effusion volume and mucosal oedema in a guinea pig model of OME Ref. , as has tro Ref. . Similartro Ref. . Metformies Ref. . AnotherERK Ref.  reduces A summary of the discussed mechanism of disease in OM subgroups is demonstrated in OM comprises a broad spectrum of interrelated pathologies. Defects in host immunity appear to play a key role in the response to initial infection and a complex range of modifying factors determine if, and which, of the subgroups subsequently emerges. At present, the limited translational literature does not reflect the substantial clinical burden of OM. Much information to date has been derived from cell lines and animal models of OM, which may not fully reflect the human pathology. Difficulties in the acquisition of human middle ear tissue have likely played a part in this and the development of physiological systems to study OM is required. If defects in mechanisms of disease could be identified, pharmacological manipulation would be an appealing alternative to existing surgical treatments; reducing both risk and cost to patients who, until now, find themselves with limited treatment options.


Nanomaterials-based phototherapies, mainly including photothermal therapy (PTT), photodynamic therapy (PDT) and photoimmunotherapy (PIT), present high efficacy, minimal invasion and negligible adverse effects in cancer treatment. The integrated phototherapeutic modalities can enhance the efficiency of cancer immunotherapy for clinical application transformation. The near-infrared (NIR) light source enables phototherapies with the high penetration depth in the biological tissues, less toxic to normal cells and tissues and a low dose of light irradiation. Mediated via the novel NIR-responsive nanomaterials, PTT and PDT are able to provoke cancer cells apoptosis from the generated heat and reactive oxygen species, respectively. The released cancer-specific antigens and membrane damage danger signals from the damaged cancer cells trigger immune responses, which would enhance the antitumor efficacy via a variety of immunotherapy. This review summarized the recent advances in NIR-triggered photo-/immune-therapeutic modalities and their synergistic mechanisms and applications toward cancers. Furthermore, the challenges, potential solutions and future directions of NIR-triggered photo-/immunotherapy were briefly discussed. Cancer is still one of the top leading causes of mortality after many decades’ search and exploration for a cure in cancer from diagnosis and therapy. Currently, the available treatments to cancers in clinics are mainly surgery, chemotherapy and radiotherapy. A number of newly developed therapeutic modalities have been selectively adopted or in preliminary clinical trials including gene therapy, targeted therapy, immunotherapy, phototherapy , magnetic hyperthermia therapy and other non-mainstream treatments.The tumor cell death are caused in virtue of extrinsic stimuli in most of the aforementioned therapeutic modalities, such as chemotherapy mediated by chemotherapeutic agents, radiotherapy via various energy rays, hyperthermia therapy from the external high-frequency magnetic field, and phototherapy attributed to external light energy sources. Photothermal therapy (PTT) and photodynamic therapy (PDT) are the two representative phototherapy and are used for the tumor cells destruction through heat or reactive oxygen species respectively under irradiation of light. The external stimuli therapeutic approaches show significant advance in ablating solid primary tumors. However, the side effects from the extrinsic stimuli are quite strong and the recurrence of cancers is not controllable. Among the extrinsic stimuli, light energy in phototherapy offers lowest side effects and least systemic toxicity. Near-infrared (NIR) light is the preferable light source due to its superior tissue penetration ability. Under light irradiation, phototherapeutic agents that are little toxic in dark are turned into functional ones to selectively destroy cancer cells, without causing much damage to surrounding healthy tissues .®) has been used to treat prostate cancer since 2010  toward cancers using nanomaterials are collected and discussed. We first describe the therapeutic effects on cancers using the synergistic PIT of immunotherapy with PDT and PTT followed with photochemistry-based cancer therapy. Lastly, a conclusion and future perspective are drawn at the end.Phototherapy is a non-invasive or minimally invasive therapeutic strategy. In cancer treatment, phototherapy not only kill tumor cells directly but also induces ICD to initiate a systemic antitumor immune response, including the redistribution and activation of immune effector cells, the expression and secretion of cytokines and the transformation of memory T lymphocytes .in situ autovaccine  that convert photon energy into thermal energy . Tumor cNIR light has been widely used as the light source for PTT exhibiting minimal absorption and scattering by tissue components, thus NIR is able to achieve deep tissue penetration. The more effective way is to integrate the two therapeutic modalities, PTT and immunotherapy, together to target the primary tumor ablation as well as the metastatic cancer cell growth. The typical immunotherapy methods have been combined with PTT via either separate but subsequent administration or single and simultaneous administration. For example, the elimination of metastatic melanoma was achieved using gold nanoshell-enabled PTT together with adoptive T cell transfer immunotherapy 24 h following PTT . While aAn immunoadjuvant agent, resiquimod R848, was loaded into the polydopamine nanoparticles as photothermal conversion agent together with carbon dots as imaging agent. Under 808 nm NIR light irradiation, the polydopamine can utilize hyperthermia to perform PTT to eliminate the primary 4T1 breast tumor cells. After the PTT, the generated and released tumor–associated antigens along with the PTT could be amplified by R848 and further triggered stronger infiltration of CTLs into distant tumors. In addition, The long-term immune memory effect was induced to prevent tumor reoccurrence .Two different immunotherapy methods can be synergized with PTT to effectively suppress primary tumors and to inhibit metastasis. Although the PTT induced immunotherapy efficacy is low and weak, the combined PTT with different immunotherapy has achieved substantial immune stimulation and overcome immunosuppression within the tumor microenvironment. More efforts are still required to explore synergistic and efficient PTT and immunotherapy.2O2 and hydroxyl and superoxide anion radicals  tumor microenvironment and generated massive oxygen which was utilized for the production of powerful ROS from the gold nanocages under the irradiation of NIR 808 nm. The primary tumor could be destroyed by these ROS, at the same time, the immunogenic cell death–mediated antitumor immune response from DAMPs was elicited to inhibitate lung metastasis  is an emerging luminescent nanomaterials with potential advantageous benefits for PDT. UCNPs are able to transfer the NIR light energy (commonly 980 or 808 nm NIR lights) into required high energy light (visible and ultraviolet lights) to excite the combined photosensitizers for the ROS generation. The limitations for the PDT exist in the biosafety concern of photosensitizers, the penetration depth of the light source (NIR light can penetrate around 1 cm penetration in tissues), and less efficacy to large solid tumors. In spite of these limitations, PDT combining with immunotherapy is still a promising approach for advanced cancer treatment . A greatOther than the two typical phototherapy modality, PTT and PDT, NIR light is also adopted to trigger antitumor immunity as photo-immunotherapy toward cancers. NIR light was used to initiate photochemistry reactions of the photosensitizer and thus induce the immunotherapy. Kobayashi’s group  proposedde novo tumor antigen-specific T-cell responses absent at baseline. The addition of PD-1 blockade reversed adaptive immune resistance, resulting in both enhanced pre-existing tumor antigen-specific T-cell responses and enhanced de novo T-cell responses induced by NIR-PIT.NIR-PIT was also combined with an immunotherapeutic method of checkpoint blockade . NIR-PITin vivo with NIR light mediated using upconversion nanoparticles (UCNPs). It was a smart system with superior spatiotemporal precision and enhanced safety. The working mechanism scheme is displayed in The photochemistry based immunotherapy shows as high potential as the PTT and PDT with less complexity in nanomaterials design and working processes. More advanced photochemistry-based immunotherapy modality should be working on.The implantation of multi-synergistic therapy relies on the combination of multiple treatments into a single nanoplatform. There are more than two phototherapeutic modalities have been integrated with immunotherapy.1O2) cleavable linker. Upon NIR laser irradiation, the SPN core within OSPS could generate both heat and 1O2 for combinational PTT and PDT of tumors, leading to the release of tumor-associated antigens. Meanwhile, the generated 1O2 specifically cleaves the 1O2-cleavable liner to trigger the on-demand release and simultaneous activation of caged NLG919, which resulted in the promoted proliferation and activation of effector T cell but suppression of Treg cells. Therefore, the synergistic phototherapy with remote-controlled immune checkpoint blockade therapy exerted an amplified therapeutic efficacy in inhibition of primary and distant tumor growth and lung metastasis  as a light absorber, rose bengal (RB) as photosensitizer and the lipid molecule  as the antigen-capturing agent. The ICG significantly enhanced the RB-based PDT efficiency of UCNP/ICG/RB-mal under NIR irradiation, simultaneously achieving selective PTT effects. Most importantly, tumor-derived antigens released from the ablated tumor could be captured and retained se model .2MoS4 nanoparticles loaded with glucose oxidase (GOx)  through the chemical reactions of the redox couples of Cu1+/2+ and Mo4+/6+. The radicals also could deplete the overexpressed glutathione to modify the tumor microenvironment aiming for the mitigation of antioxidant capability of the tumors.A cascade bioreactor for synergistic cancer therapy combining chemo-dynamic therapy (CDT)/starvation therapy/phototherapy/immunotherapy was developed based on hollow structured Cuse (GOx) . The hol2MoS4 could catalyze the reaction of endogenous H2O2 to O2 which would react with glucose and produce H2O2 again under the catalysis of GOx. The consummation of glucose could shut down nutrient supply to cancer cells and achieve starvation therapy. At the same time, the phototherapy effect of the Cu2MoS4 under 1064 nm NIR light irradiation was demonstrated by the strong tumor-killing capacity due to its excellent photothermal conversion efficiency. More importantly, the PEGylated Cu2MoS4@GOx-based synergistic therapy combined with checkpoint blockade therapy could elicite robust immune responses for both effectively ablating primary tumors and inhibiting cancer metastasis.Furthermore, the CuThe aforementioned photo/immunotherapy are summarized in in vivo microenvironment, the performance of photoimmunotherapy in human is barely predicted. For example, Challenges and promising solutions are ahead of the success of photo/immunotherapy in cancer treatment. One of the major challenges is the lack of clinical trial results. Preclinical research in animals has shown that the photoimmunotherapy can eliminate tumor cells and enhance tumor-cell-selective systemic host-immunity leading to boosted antitumor effects triggered by NIR light. However, due to the species difference and complexity of human Functional targeting moieties and targeting efficiency is another critical factor that would influence the localized treatment. The targeting moieties bind surface markers on the cancer cells, dominating the specificity and distribution of the photoimmunotherapy agents. Various targeting moieties including antibodies, peptides, functional molecules and oligonucleotides have been conjugated with the photoactivation complexes . More spThe nanomaterials provide a large pool of candidates as the photoactivated cores for photoimmunotherapy. The development of nanomaterials with low cytotoxicity, high photoactivity, and multifunctionality will facilitate the clinical application of cancer photoimmunotherapy. Proper NIR light is required for deep penetration into the tumour tissues, dependent on the nanomaterials applied in the therapy. The energy of NIR light dramatically decreases with the depth of the tissue, for example, NIR photoimmunotherapy through bone is limited to 3 mm when using 810 nm NIR of less than 1 W . ImproviWell-integrated photo/immunotherapeutic modalities will benefit the single administration and working simultaneously. PTT and PDT, in combination with immunotherapies, provide infinite possibilities as a promising method to treat cancer. The administration or conjugation of antibodies or gene silencing agents aiming to immune checkpoint pathways with PTT and PDT open new windows for immunotherapy . HoweverThe ultimate goal of the nanomaterials-based phototherapies is to eradicate cancers completely, including primary solid tumors and metastatic cancers. With minimized side effect and maximized treatment efficacy, phototherapy and immunotherapy are the two most promising therapeutic modalities and their synergistic combination of photoimmunotherapy has shown substantial potentials to realize the ultimate goal.XX designed, structured, and wrote the manuscript. HL designed, structured, and revised the manuscript. RL designed andrevised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


As a very common risk factor of cardiovascular disease (CVD) and premature death, hypertension is preventable, and extensively controlling blood pressure to targets set forth by the current guideline is key to prevent and manage the debilitating disorder and CVD. In recent decades, the advancement of multiomics studies including genomics, epigenomics, transcriptomics, proteomics, metabolomics, and gut microbiome has uncovered many potential biomarkers for hypertension and its related cardiovascular complications including myocardial infarction, heart failure, and stroke –3. Due tThe paper written by Dr. X. Peng et al. reported a longitudinal association between dynamic fasting glucose and risk of incident stroke in 12,321 Chinese adults. They found that a higher variance in addition to a higher level of fasting glucose predicted a higher risk of incident stroke, independent of lifestyle behaviors and metabolic factors. These results suggest that high variability of fasting glucose may be a risk factor for stroke. The homeostasis of glucose metabolism should be maintained for community members, diabetic patients in particular.In the paper written by Dr. M.-C. Chen et al., the authors examined whether serum angiopoietin-like protein 3 (ANGPTL3) levels at baseline could predict adverse cardiovascular events in 90 patients with coronary artery disease (CAD). After a median follow-up of 54 months, 33 patients developed major adverse cardiovascular events, and these patients had higher ANGPTL3 levels at baseline compared with those who were free of adverse cardiovascular events during the follow-up. Cox regression models revealed that a higher baseline level of ANGPTL3 was significantly associated with an increased risk of developing adverse cardiovascular events for CAD patients. ANGPTL3 may be a predictor for major adverse cardiovascular events in CAD patients, but the efficacy and effectiveness of screening ANGPTL3 for CAD patients warrant further evaluation.Leveraging data from 285 ischemic stroke patients receiving intravenous r-tPA thrombolysis within 4.5 hours of stroke onset, Dr. Y.-L. Liu and colleagues found that higher neutrophil-to-lymphocyte ratio at admission predicted risks of hemorrhagic transformation after r-tPA thrombolysis in acute ischemic stroke patients. Neutrophil-to-lymphocyte ratio, a parameter that can be easily estimated from blood cell count analysis, could be a useful marker for predicting hemorrhagic transformation in acute ischemic stroke patients after intravenous r-tPA thrombolysis.The paper written by Dr. J. Zhang and colleagues reported a modification effect of platelet count on the association between homocysteine and blood pressure in more than 30,000 hypertensive patients. The association between homocysteine and blood pressure was much stronger in participants having low platelet count, compared with those having high platelet count. These results indicate that platelet count may be useful in the identification of individuals who are major beneficiaries of reducing-homocysteine treatments, but the clinical application still have a long way to go.Dr. H. Xie et al. conducted a nested case-control study to examine the association between plasma urotensin II and the risks of incident hypertension by exploring 723 participants who developed hypertension and 1,096 participants who remained free of hypertension after about 5 years of follow-up. In contrast to prior case-control studies, they did not observe a significant association between urotensin II and hypertension. The causal effect of urotensin II on hypertension is still unspecified.Dr. X. Jia et al. conducted an animal study to test the effect of resveratrol on pregnant hypertension reduction using mice models of high-salt diet-induced hypertension. They found that resveratrol supplementation could decrease blood pressure, serum urea, serum creatinine, and urinary protein in hypertensive pregnant mice. Resveratrol could be a promising candidate of an effective blood pressure lowering agent for pregnancy-induced hypertension, but further evidence is needed from clinical trials in humans.Learning the new biomarkers of hypertension may help clinicians and researchers to understand the underlying mechanisms and provide additional motivation for undertaking the difficult challenge to reduce hypertension and its related cardiovascular disorders.