Signaling through the type 2 cannabinoid receptor regulates the severity of acute and chronic graft-versus-host disease.

Graft-versus-host disease (GVHD) pathophysiology is a complex interplay between cells that comprise the adaptive and innate arms of the immune system. Effective prophylactic strategies are therefore contingent upon approaches that address contributions from both immune cell compartments. In the current study, we examined the role of the type 2 cannabinoid receptor (CB2R), which is expressed on nearly all immune cells, and demonstrated that absence of the CB2R on donor CD4+ or CD8+ T cells or administration of a selective CB2R pharmacological antagonist exacerbated acute GVHD lethality. This was accompanied primarily by the expansion of proinflammatory CD8+ T cells, indicating that constitutive CB2R expression on T cells preferentially regulated CD8+ T-cell alloreactivity. Using a novel CB2ReGFP reporter mouse, we observed significant loss of CB2R expression on T cells, but not macrophages, during acute GVHD, indicative of differential alterations in receptor expression under inflammatory conditions. Therapeutic targeting of the CB2R with the agonists Δ9-tetrahydrocannabinol (THC) and JWH-133 revealed that only THC mitigated lethal T cell-mediated acute GVHD. Conversely, only JWH-133 was effective in a sclerodermatous chronic GVHD model where macrophages contributed to disease biology. In vitro, both THC and JWH-133 induced arrestin recruitment and extracellular regulated kinase phosphorylation via CB2R, but THC had no effect on CB2R-mediated inhibition of adenylyl cyclase. This study shows that the CB2R plays a critical role in the regulation of GVHD and suggests that effective therapeutic targeting is dependent upon agonist signaling characteristics and receptor selectivity in conjunction with the composition of pathogenic immune effector cells.

IL6Myc mouse is an immunocompetent model for the development of aggressive multiple myeloma.

Multiple Myeloma (MM) is a plasma cell neoplasm originating in the bone marrow and is the second most common blood cancer in the United States. One challenge in understanding the pathogenesis of MM and improving treatment is a lack of immunocompetent mouse models. We previously developed the IL6Myc mouse that generates plasmacytomas at 100% penetrance that phenotypically resemble aggressive MM. Using comprehensive genomic analysis, we found that the IL6Myc tumors resemble aggressive MM by RNA and protein expression. We also found that IL6Myc tumors accumulated fusions and missense mutations in genes that overlap significantly with human myeloma, indicating that the mouse is good model for studying disease etiology. Lastly, we derived cell lines from IL6Myc tumors that express cell surface markers typical of MM and readily engraft into mice, home to the bone marrow, and induce osteolytic disease. The cell lines may be useful in developing immunotherapies directed against BAFF-R and TACI, though not BCMA, and may also be a good model for studying dexamethasone resistance. These data indicate that the IL6Myc model is useful for studying development of aggressive MM and for developing new treatments against such forms of the disease.

Pathogenic Bhlhe40+ GM-CSF+ CD4+ T cells promote indirect alloantigen presentation in the GI tract during GVHD.

Gastrointestinal (GI) tract involvement is the major cause of morbidity and mortality in acute graft-versus-host disease (GVHD), and pathological damage is largely attributable to inflammatory cytokine production. Recently, granulocyte-macrophage colony stimulating factor (GM-CSF) has been identified as a cytokine that mediates inflammation in the GI tract, but the transcriptional program that governs GM-CSF production and the mechanism by which GM-CSF links adaptive to innate immunity within this tissue site have not been defined. In the current study, we identified Bhlhe40 as a key transcriptional regulator that governs GM-CSF production by CD4+ T cells and mediates pathological damage in the GI tract during GVHD. In addition, we observed that GM-CSF was not regulated by either interleukin 6 (IL-6) or IL-23, which are both potent inducers of GVHD-induced colonic pathology, indicating that GM-CSF constitutes a nonredundant inflammatory pathway in the GI tract. Mechanistically, GM-CSF had no adverse effect on regulatory T-cell reconstitution, but linked adaptive to innate immunity by enhancing the activation of donor-derived dendritic cells in the colon and subsequent accumulation of these cells in the mLNs. In addition, GM-CSF promoted indirect alloantigen presentation, resulting in the accumulation of donor-derived T cells with a proinflammatory cytokine phenotype in the colon. Thus, Bhlhe40+ GM-CSF+ CD4+ T cells constitute a colitogenic T-cell population that promotes indirect alloantigen presentation and pathological damage within the GI tract, positioning GM-CSF as a key regulator of GVHD in the colon and a potential therapeutic target for amelioration of this disease.

Kras-Deficient T Cells Attenuate Graft-versus-Host Disease but Retain Graft-versus-Leukemia Activity.

Acute graft-versus-host disease (aGVHD) is one major serious complication that is induced by alloreactive donor T cells recognizing host Ags and limits the success of allogeneic hematopoietic stem cell transplantation. In the current studies, we identified a critical role of Kras in regulating alloreactive T cell function during aGVHD. Kras deletion in donor T cells dramatically reduced aGVHD mortality and severity in an MHC-mismatched allogeneic hematopoietic stem cell transplantation mouse model but largely maintained the antitumor capacity. Kras-deficient CD4 and CD8 T cells exhibited impaired TCR-induced activation of the ERK pathway. Kras deficiency altered TCR-induced gene expression profiles, including the reduced expression of various inflammatory cytokines and chemokines. Moreover, Kras deficiency inhibited IL-6-mediated Th17 cell differentiation and impaired IL-6-induced ERK activation and gene expression in CD4 T cells. These findings support Kras as a novel and effective therapeutic target for aGVHD.

Bim regulates the survival and suppressive capability of CD8+ FOXP3+ regulatory T cells during murine GVHD.

CD8+ Foxp3+ T cells (Tregs) are a potent regulatory population whose functional and ontological similarities to CD4+ Fox3+ T cells have not been well delineated. Using an experimental model of graft-versus-host disease (GVHD), we observed that CD8+ Tregs were significantly less potent than CD4+ Tregs for the suppression of GVHD. To define the mechanistic basis for this observation, we examined the T-cell repertoire and the transcriptional profile of in vivo-derived CD4+ and CD8+ Tregs that emerged early during this disease. Polyclonal and alloantigen-induced CD8+ Tregs had repertoire diversity that was similar to that of conventional CD8+ T cells, indicating that a restricted repertoire was not the proximate cause of decreased suppression. Transcriptional profiling revealed that CD8+ Tregs possessed a canonical Treg transcriptional signature that was similar to that observed in CD4+ Tregs, yet distinct from conventional CD8+ T cells. Pathway analysis, however, demonstrated that CD8+ Tregs had differential gene expression in pathways involved in cell death and survival. This was further confirmed by detailed mRNA sequence analysis and protein expression studies, which demonstrated that CD8+ Tregs had increased expression of Bim and reduced expression of Mcl-1. Transplantation with CD8+ Foxp3+ Bim-/- Tregs resulted in prolonged Treg survival and reduced GVHD lethality compared with wild-type CD8+ Tregs, providing functional confirmation that increased expression of Bim was responsible for reduced in vivo efficacy. Thus, Bim regulates the survival and suppressive capability of CD8+ Tregs, which may have implications for their use in regulatory T-cell therapy.

Blockade of interleukin-27 signaling reduces GVHD in mice by augmenting Treg reconstitution and stabilizing Foxp3 expression.

Reestablishment of competent regulatory pathways has emerged as a strategy to reduce the severity of graft-versus-host disease (GVHD), and recalibrate the effector and regulatory arms of the immune system. However, clinically feasible, cost-effective strategies that do not require extensive ex vivo cellular manipulation have remained elusive. In the current study, we demonstrate that inhibition of the interleukin-27p28 (IL-27p28) signaling pathway through antibody blockade or genetic ablation prevented lethal GVHD in multiple murine transplant models. Moreover, protection from GVHD was attributable to augmented global reconstitution of CD4+ natural regulatory T cells (nTregs), CD4+ induced Tregs (iTregs), and CD8+ iTregs, and was more potent than temporally concordant blockade of IL-6 signaling. Inhibition of IL-27p28 also enhanced the suppressive capacity of adoptively transferred CD4+ nTregs by increasing the stability of Foxp3 expression. Notably, blockade of IL-27p28 signaling reduced T-cell-derived-IL-10 production in conventional T cells; however, there was no corresponding effect in CD4+ or CD8+ Tregs, indicating that IL-27 inhibition had differential effects on IL-10 production and preserved a mechanistic pathway by which Tregs are known to suppress GVHD. Targeting of IL-27 therefore represents a novel strategy for the in vivo expansion of Tregs and subsequent prevention of GVHD without the requirement for ex vivo cellular manipulation, and provides additional support for the critical proinflammatory role that members of the IL-6 and IL-12 cytokine families play in GVHD biology.

Development and internal validation of the multivariable CIPHER (Collaborative Integrated Pregnancy High-dependency Estimate of Risk) clinical risk prediction model.

BACKGROUND: Intensive care unit (ICU) outcome prediction models, such as Acute Physiology And Chronic Health Evaluation (APACHE), were designed in general critical care populations and their use in obstetric populations is contentious. The aim of the CIPHER (Collaborative Integrated Pregnancy High-dependency Estimate of Risk) study was to develop and internally validate a multivariable prognostic model calibrated specifically for pregnant or recently delivered women admitted for critical care. METHODS: A retrospective observational cohort was created for this study from 13 tertiary facilities across five high-income and six low- or middle-income countries. Women admitted to an ICU for more than 24 h during pregnancy or less than 6 weeks post-partum from 2000 to 2012 were included in the cohort. A composite primary outcome was defined as maternal death or need for organ support for more than 7 days or acute life-saving intervention. Model development involved selection of candidate predictor variables based on prior evidence of effect, availability across study sites, and use of LASSO (Least Absolute Shrinkage and Selection Operator) model building after multiple imputation using chained equations to address missing data for variable selection. The final model was estimated using multivariable logistic regression. Internal validation was completed using bootstrapping to correct for optimism in model performance measures of discrimination and calibration. RESULTS: Overall, 127 out of 769 (16.5%) women experienced an adverse outcome. Predictors included in the final CIPHER model were maternal age, surgery in the preceding 24 h, systolic blood pressure, Glasgow Coma Scale score, serum sodium, serum potassium, activated partial thromboplastin time, arterial blood gas (ABG) pH, serum creatinine, and serum bilirubin. After internal validation, the model maintained excellent discrimination (area under the curve of the receiver operating characteristic (AUROC) 0.82, 95% confidence interval (CI) 0.81 to 0.84) and good calibration (slope of 0.92, 95% CI 0.91 to 0.92 and intercept of -0.11, 95% CI -0.13 to -0.08). CONCLUSIONS: The CIPHER model has the potential to be a pragmatic risk prediction tool. CIPHER can identify critically ill pregnant women at highest risk for adverse outcomes, inform counseling of patients about risk, and facilitate bench-marking of outcomes between centers by adjusting for baseline risk.

Physiological ranges of matrix rigidity modulate primary mouse hepatocyte function in part through hepatocyte nuclear factor 4 alpha.

UNLABELLED: Matrix rigidity has important effects on cell behavior and is increased during liver fibrosis; however, its effect on primary hepatocyte function is unknown. We hypothesized that increased matrix rigidity in fibrotic livers would activate mechanotransduction in hepatocytes and lead to inhibition of liver-specific functions. To determine the physiologically relevant ranges of matrix stiffness at the cellular level, we performed detailed atomic force microscopy analysis across liver lobules from normal and fibrotic livers. We determined that normal liver matrix stiffness was around 150 Pa and increased to 1-6 kPa in areas near fibrillar collagen deposition in fibrotic livers. In vitro culture of primary hepatocytes on collagen matrix of tunable rigidity demonstrated that fibrotic levels of matrix stiffness had profound effects on cytoskeletal tension and significantly inhibited hepatocyte-specific functions. Normal liver stiffness maintained functional gene regulation by hepatocyte nuclear factor 4 alpha (HNF4α), whereas fibrotic matrix stiffness inhibited the HNF4α transcriptional network. Fibrotic levels of matrix stiffness activated mechanotransduction in primary hepatocytes through focal adhesion kinase. In addition, blockade of the Rho/Rho-associated protein kinase pathway rescued HNF4α expression from hepatocytes cultured on stiff matrix. CONCLUSION: Fibrotic levels of matrix stiffness significantly inhibit hepatocyte-specific functions in part by inhibiting the HNF4α transcriptional network mediated through the Rho/Rho-associated protein kinase pathway. Increased appreciation of the role of matrix rigidity in modulating hepatocyte function will advance our understanding of the mechanisms of hepatocyte dysfunction in liver cirrhosis and spur development of novel treatments for chronic liver disease. (Hepatology 2016;64:261-275).

Direct orthotopic implantation of hepatic organoids.

BACKGROUND: Liver organoids show potential for development as a tissue replacement therapy for patients with end-stage liver disease, but efficient methods for introducing organoids into host livers have not been established. In this study, we aimed to develop a surgical technique to implant hepatic organoids into the liver and assess their engraftment. METHODS: Donor hepatocytes were isolated from ROSA26 C57BL/6 mice, so that engrafted cells, when implanted into wild-type mice, could be easily identified by X-gal staining. Hepatic organoids were generated by three-dimensional culture in rotating wall vessel bioreactors. We qualitatively and quantitatively compared organoid engraftment to that of single-cell hepatocyte transplants. In addition, we determined the effect of adding stellate cells to hepatocytes to form co-aggregated organoids and the effect of partial hepatectomy of the host liver on organoid engraftment. RESULTS: Direct orthotopic implantation of hepatic organoids within a hepatotomy site resulted in local engraftment of exogenous hepatocytes with limited durability. Hepatocyte-stellate cell organoids produced more extracellular matrix but did not significantly improve engraftment compared with hepatocyte-alone organoids. Partial hepatectomy of the host liver led to significantly decreased engraftment of organoids. Survival of organoids was limited by the presence of apoptotic hepatocytes within organoids as early as 1 h after implantation. Organoids eventually became necrotic and elicited a chronic inflammatory giant cell reaction similar to a foreign body response. CONCLUSIONS: With additional organoid and host factor optimization, direct orthotopic implantation of hepatic organoids may be an approach to introduce large numbers of exogenous hepatocytes into recipient livers.

Posterior parietal cortex is critical for the encoding, consolidation, and retrieval of a memory that guides attention for learning.

Within most contemporary learning theories, reinforcement prediction error, the difference between the obtained and expected reinforcer value, critically influences associative learning. In some theories, this prediction error determines the momentary effectiveness of the reinforcer itself, such that the same physical event produces more learning when its presentation is surprising than when it is expected. In other theories, prediction error enhances attention to potential cues for that reinforcer by adjusting cue-specific associability parameters, biasing the processing of those stimuli so that they more readily enter into new associations in the future. A unique feature of these latter theories is that such alterations in stimulus associability must be represented in memory in an enduring fashion. Indeed, considerable data indicate that altered associability may be expressed days after its induction. Previous research from our laboratory identified brain circuit elements critical to the enhancement of stimulus associability by the omission of an expected event, and to the subsequent expression of that altered associability in more rapid learning. Here, for the first time, we identified a brain region, the posterior parietal cortex, as a potential site for a memorial representation of altered stimulus associability. In three experiments using rats and a serial prediction task, we found that intact posterior parietal cortex function was essential during the encoding, consolidation, and retrieval of an associability memory enhanced by surprising omissions. We discuss these new results in the context of our previous findings and additional plausible frontoparietal and subcortical networks.

Blocking in autoshaped lever-pressing procedures with rats.

Rats will approach and contact a lever whose insertion into the chamber signals response-independent food delivery. This "autoshaping" or "sign-tracking" phenomenon has recently attracted considerable attention as a platform for studying individual differences in impulsivity, drug sensitization, and other traits associated with vulnerability to drug addiction. Here, we examined two basic stimulus selection phenomena-blocking and overshadowing-in the autoshaped lever pressing of rats. Blocking and overshadowing were decidedly asymmetrical. Previously reinforced lever-extension conditioned stimuli (CSs) completely blocked conditioning to auditory cues (Exps. 1 and 2), and previously nonreinforced lever-extension CSs overshadowed conditioning to auditory cues. By contrast, conditioning to lever-extension CSs was not blocked by either auditory (Exp. 3) or lever-insertion (Exp. 4) cues, and was not overshadowed by auditory cues. Conditioning to a lever-insertion cue was somewhat overshadowed by the presence of another lever, especially in terms of food cup behavior displayed after lever withdrawal. We discuss several frameworks in which the apparent immunity of autoshaped lever pressing to blocking might be understood. Given evidence that different brain systems are engaged when different kinds of cues are paired with food delivery, it is worth considering the possibility that interactions among them in learning and performance may follow different rules. In particular, it is intriguing to speculate that the roles of simple cue-reinforcer contiguity, as well as of individual and aggregate reinforcer prediction errors, may differ across stimulus classes.

Risk of Sudden Death in Patients With RASopathy Hypertrophic Cardiomyopathy.

BACKGROUND: Genetic defects in the RAS/mitogen-activated protein kinase pathway are an important cause of hypertrophic cardiomyopathy (RAS-HCM). Unlike primary HCM (P-HCM), the risk of sudden cardiac death (SCD) and long-term survival in RAS-HCM are poorly understood. OBJECTIVES: The study's objective was to compare transplant-free survival, incidence of SCD, and implantable cardioverter-defibrillator (ICD) use between RAS-HCM and P-HCM patients. METHODS: In an international, 21-center cohort study, we analyzed phenotype-positive pediatric RAS-HCM (n = 188) and P-HCM (n = 567) patients. The between-group differences in cumulative incidence of all outcomes from first evaluation were compared using Gray's tests, and age-related hazard of all-cause mortality was determined. RESULTS: RAS-HCM patients had a lower median age at diagnosis compared to P-HCM (0.9 years [IQR: 0.2-5.0 years] vs 9.8 years [IQR: 2.0-13.9 years], respectively) (P < 0.001). The 10-year cumulative incidence of SCD from first evaluation was not different between RAS-HCM and P-HCM (4.7% vs 4.2%, respectively; P = 0.59). The 10-year cumulative incidence of nonarrhythmic deaths or transplant was higher in RAS-HCM compared with P-HCM (11.0% vs 5.4%, respectively; P = 0.011). The 10-year cumulative incidence of ICD insertions, however, was 5-fold lower in RAS-HCM compared with P-HCM (6.9% vs 36.6%; P < 0.001). Nonarrhythmic deaths occurred primarily in infancy and SCD primarily in adolescence. CONCLUSIONS: RAS-HCM was associated with a higher incidence of nonarrhythmic death or transplant but similar incidence of SCD as P-HCM. However, ICDs were used less frequently in RAS-HCM compared to P-HCM. In addition to monitoring for heart failure and timely consideration of advanced heart failure therapies, better risk stratification is needed to guide ICD practices in RAS-HCM.

MRI Insights Into Adolescent Neurocircuitry-A Vision for the Future.

Adolescence is the time of onset of many psychiatric disorders. Half of pediatric patients present with comorbid psychiatric disorders that complicate both their medical and psychiatric care. Currently, diagnosis and treatment decisions are based on symptoms. The field urgently needs brain-based diagnosis and personalized care. Neuroimaging can shed light on how aberrations in brain circuits might underlie psychiatric disorders and their development in adolescents. In this perspective article, we summarize recent MRI literature that provides insights into development of psychiatric disorders in adolescents. We specifically focus on studies of brain structural and functional connectivity. Ninety-six included studies demonstrate the potential of MRI to assess psychiatrically relevant constructs, diagnose psychiatric disorders, predict their development or predict response to treatment. Limitations of the included studies are discussed, and recommendations for future research are offered. We also present a vision for the role that neuroimaging may play in pediatrics and primary care in the future: a routine neuropsychological and neuropsychiatric imaging (NPPI) protocol for adolescent patients, which would include a 30-min brain scan, a quality control and safety read of the scan, followed by computer-based calculation of the structural and functional brain network metrics that can be compared to the normative data by the pediatrician. We also perform a cost-benefit analysis to support this vision and provide a roadmap of the steps required for this vision to be implemented.

Liver epithelial focal adhesion kinase modulates fibrogenesis and hedgehog signaling.

Focal adhesion kinase (FAK) is an important mediator of extracellular matrix-integrin mechano-signal transduction that regulates cell motility, survival, and proliferation. As such, FAK is being investigated as a potential therapeutic target for malignant and fibrotic diseases, and numerous clinical trials of FAK inhibitors are underway. The function of FAK in nonmalignant, nonmotile epithelial cells is not well understood. We previously showed that hepatocytes demonstrated activated FAK near stiff collagen tracts in fibrotic livers. In this study, we examined the role of liver epithelial FAK by inducing fibrotic liver disease in mice with liver epithelial FAK deficiency. We found that mice that lacked FAK in liver epithelial cells developed more severe liver injury and worse fibrosis as compared with controls. Increased fibrosis in liver epithelial FAK-deficient mice was linked to the activation of several profibrotic pathways, including the hedgehog/smoothened pathway. FAK-deficient hepatocytes produced increased Indian hedgehog in a manner dependent on matrix stiffness. Furthermore, expression of the hedgehog receptor, smoothened, was increased in macrophages and biliary cells of hepatocyte-specific FAK-deficient fibrotic livers. These results indicate that liver epithelial FAK has important regulatory roles in the response to liver injury and progression of fibrosis.

Using non-thermal irreversible electroporation to create an in vivo niche for exogenous cell engraftment.

The critical shortage of donor organs has spurred investigation of alternative approaches to either generate replacement organs or implant exogenous cells for treatment of end-stage organ failure. Non-thermal irreversible electroporation (NTIRE), which uses brief high electric field pulses to induce irreversible permeabilization of cell membranes, has emerged as a technique for tumor ablation. Here, we introduce a new application for NTIRE that employs in situ cell ablation to create a niche within a solid organ for engraftment of exogenous cells in vivo. We treated the livers of mice with NTIRE and subsequently implanted exogenous congenic hepatocytes within the zone of cell ablation. Donor hepatocytes engrafted and integrated with host liver parenchyma pre-treated with NTIRE. This new approach should have value for studying the effects of a native matrix scaffold on in vivo cell growth and may pioneer a new type of minimally-invasive regenerative surgery.

Host interleukin 6 production regulates inflammation but not tryptophan metabolism in the brain during murine GVHD.

Graft-versus-host disease (GVHD) induces pathological damage in peripheral target organs leading to well-characterized, organ-specific clinical manifestations. Patients with GVHD, however, can also have behavioral alterations that affect overall cognitive function, but the extent to which GVHD alters inflammatory and biochemical pathways in the brain remain poorly understood. In the current study, we employed complementary murine GVHD models to demonstrate that alloreactive donor T cells accumulate in the brain and affect a proinflammatory cytokine milieu that is associated with specific behavioral abnormalities. Host IL-6 was identified as a pivotal cytokine mediator, as was host indoleamine 2,3-dioxygenase (IDO-1), which was upregulated in GVHD in an IL-6-dependent manner in microglial cells and was accompanied by dysregulated tryptophan metabolism in the dorsal raphe nucleus and prefrontal cortex. Blockade of the IL-6 signaling pathway significantly reduced donor T cell accumulation, inflammatory cytokine gene expression, and host microglial cell expansion, but did not reverse GVHD-induced tryptophan metabolite dysregulation. Thus, these results indicate that inhibition of IL-6 signaling attenuates neuroinflammation, but does not reverse all of the metabolic abnormalities in the brain during GVHD, which may also have implications for the treatment of neurotoxicity occurring after other T cell-based immune therapies with IL-6-directed approaches.

A colitogenic memory CD4+ T cell population mediates gastrointestinal graft-versus-host disease.

Damage to the gastrointestinal tract is a major cause of morbidity and mortality in graft-versus-host disease (GVHD) and is attributable to T cell-mediated inflammation. In this work, we identified a unique CD4+ T cell population that constitutively expresses the ß2 integrin CD11c and displays a biased central memory phenotype and memory T cell transcriptional profile, innate-like properties, and increased expression of the gut-homing molecules α4ß7 and CCR9. Using several complementary murine GVHD models, we determined that adoptive transfer and early accumulation of ß2 integrin-expressing CD4+ T cells in the gastrointestinal tract initiated Th1-mediated proinflammatory cytokine production, augmented pathological damage in the colon, and increased mortality. The pathogenic effect of this CD4+ T cell population critically depended on coexpression of the IL-23 receptor, which was required for maximal inflammatory effects. Non-Foxp3-expressing CD4+ T cells produced IL-10, which regulated colonic inflammation and attenuated lethality in the absence of functional CD4+Foxp3+ T cells. Thus, the coordinate expression of CD11c and the IL-23 receptor defines an IL-10-regulated, colitogenic memory CD4+ T cell subset that is poised to initiate inflammation when there is loss of tolerance and breakdown of mucosal barriers.

The functional CD8 T cell memory recall repertoire responding to the influenza A M1(58-66) epitope is polyclonal and shows a complex clonotype distribution.

The CD8 memory T cell repertoire to the influenza A derived M1(58-66) epitope shows a restricted V genes and CDR3 sequences usage. The repertoire is highly polyclonal and the clonotype distribution has been described as consisting of two components, one showing a power law-like distribution and the other composed of a few clonotypes with a very high relative frequency. The question is whether the complex repertoire defined by its ability to flourish in a short term recall culture corresponded to functional cells. Here we show that there is a relation between expression of the degranulation marker CD107 and cytotoxicity or IFN-γ production in CD8 T cell lines and clones. We then examine recently degranulated CD8 cells from recall cultures from four middle aged HLA-A2 subjects and show that these functional cells are polyclonal. The clonotype distributions of the CD8(+)CD107(+) repertoires are complex in the same manner as previously reported. The clonotype composition of CD8(+)CD107(+) repertoires is also very similar to CD8 only repertoires, and to CD8(+)HLA-A2-M1(58-66) pentamer positive repertoires. We postulate that multiple exposures during childhood to this conserved influenza A epitope has generated a complex functional repertoire in HLA-A2 individuals.