Selected MicroRNAs Define Cell Fate Determination of Murine Central Memory CD8 T Cells

During an immune response T cells enter memory fate determination, a program that divides them into two main populations: effector memory and central memory T cells. Since in many systems protection appears to be preferentially mediated by T cells of the central memory it is important to understand when and how fate determination takes place. To date, cell intrinsic molecular events that determine their differentiation remains unclear. MicroRNAs are a class of small, evolutionarily conserved RNA molecules that negatively regulate gene expression, causing translational repression and/or messenger RNA degradation. Here, using an in vitro system where activated CD8 T cells driven by IL-2 or IL-15 become either effector memory or central memory cells, we assessed the role of microRNAs in memory T cell fate determination. We found that fate determination to central memory T cells is under the balancing effects of a discrete number of microRNAs including miR-150, miR-155 and the let-7 family. Based on miR-150 a new target, KChIP.1 (K + channel interacting protein 1), was uncovered, which is specifically upregulated in developing central memory CD8 T cells. Our studies indicate that cell fate determination such as surface phenotype and self-renewal may be decided at the pre-effector stage on the basis of the balancing effects of a discrete number of microRNAs. These results may have implications for the development of T cell vaccines and T cell-based adoptive therapies

Viral persistence redirects CD4 T cell differentiation toward T follicular helper cells

Persistent virus infection drives follicular T helper cell differentiation

Stochastic Models of Lymphocyte Proliferation and Death

Quantitative understanding of the kinetics of lymphocyte proliferation and death upon activation with an antigen is crucial for elucidating factors determining the magnitude, duration and efficiency of the immune response. Recent advances in quantitative experimental techniques, in particular intracellular labeling and multi-channel flow cytometry, allow one to measure the population structure of proliferating and dying lymphocytes for several generations with high precision. These new experimental techniques require novel quantitative methods of analysis. We review several recent mathematical approaches used to describe and analyze cell proliferation data. Using a rigorous mathematical framework, we show that two commonly used models that are based on the theories of age-structured cell populations and of branching processes, are mathematically identical. We provide several simple analytical solutions for a model in which the distribution of inter-division times follows a gamma distribution and show that this model can fit both simulated and experimental data. We also show that the estimates of some critical kinetic parameters, such as the average inter-division time, obtained by fitting models to data may depend on the assumed distribution of inter-division times, highlighting the challenges in quantitative understanding of cell kinetics

Increased percentage of T cells with the expression of CD127 and CD132 in hypertrophic adenoid in children with otitis media with effusion

The hypertrophic adenoid may promote chronic suppurative otitis media in children as it fulfills its immune function. The number of lymphocytes in the adenoid and their cooperation in the immune response depend of on their proliferation and migration to the effector sites. Interleukin 7 (IL-7) is essential for the normal development and function lymphocytes. IL-7 plays pivotal role for activation and proliferation of T and B cells. The heterodimeric interleukin-7 receptor (IL-7R) is composed of the IL-7Rα (127) and the common cytokine receptor γc (CD132). The aim of this study was to evaluate the percentage of lymphocytes T (CD4+ and CD8+) with IL-7R (CD127 and CD132) expression in hypertrophic adenoid in children suffering with otitis media with effusion for a duration of 3 months. Adenoid excised due to hypertrophy with or without chronic otitis media with effusion was used as study material. CD4+ CD127+, CD4+132+, CD8+CD127+ and CD8+CD132+ cell subpopulations were identified using monoclonal antibodies and flow cytometry. The percentage of CD4+ and CD8+ T cells with CD127 receptor expression in hypertrophic adenoid of children with otitis media with effusion was statistically significantly higher than in hypertrophic adenoid group. The percentage of CD4+ T cells with CD132 expression in the study group was statistically significantly higher than in the reference group. The percentage of CD8+ T cells with CD132+ expression was not statistically different in both groups. The increased percentage of T lymphocytes with IL-7R expression (CD127 and CD132) in hypertrophic adenoid seems to influence the quantity of lymphocytes and upset the immunological function of tonsils which can influence the course of otitis media with effusion

Imaging Trans-Cellular Neurexin-Neuroligin Interactions by Enzymatic Probe Ligation

Neurexin and neuroligin are transmembrane adhesion proteins that play an important role in organizing the neuronal synaptic cleft. Our lab previously reported a method for imaging the trans-synaptic binding of neurexin and neuroligin called BLINC (Biotin Labeling of INtercellular Contacts). In BLINC, biotin ligase (BirA) is fused to one protein while its 15-amino acid acceptor peptide substrate (AP) is fused to the binding partner. When the two fusion proteins interact across cellular junctions, BirA catalyzes the site-specific biotinylation of AP, which can be read out by staining with streptavidin-fluorophore conjugates. Here, we report that BLINC in neurons cannot be reproduced using the reporter constructs and labeling protocol previously described. We uncover the technical reasons for the lack of reproducibilty and then re-design the BLINC reporters and labeling protocol to achieve neurexin-neuroligin BLINC imaging in neuron cultures. In addition, we introduce a new method, based on lipoic acid ligase instead of biotin ligase, to image trans-cellular neurexin-neuroligin interactions in human embryonic kidney cells and in neuron cultures. This method, called ID-PRIME for Interaction-Dependent PRobe Incorporation Mediated by Enzymes, is more robust than BLINC due to higher surface expression of lipoic acid ligase fusion constructs, gives stronger and more localized labeling, and is more versatile than BLINC in terms of signal readout. ID-PRIME expands the toolkit of methods available to study trans-cellular protein-protein interactions in living systems.National Institutes of Health (U.S.) (DP1 OD003961

Enforced PGC-1α expression promotes CD8 T cell fitness, memory formation and antitumor immunity.

Memory CD8 T cells can provide long-term protection against tumors, which depends on their enhanced proliferative capacity, self-renewal and unique metabolic rewiring to sustain cellular fitness. Specifically, memory CD8 T cells engage oxidative phosphorylation and fatty acid oxidation to fulfill their metabolic demands. In contrast, tumor-infiltrating lymphocytes (TILs) display severe metabolic defects, which may underlie their functional decline. Here, we show that overexpression of proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), the master regulator of mitochondrial biogenesis (MB), favors CD8 T cell central memory formation rather than resident memory generation. PGC-1α-overexpressing CD8 T cells persist and mediate more robust recall responses to bacterial infection or peptide vaccination. Importantly, CD8 T cells with enhanced PGC-1α expression provide stronger antitumor immunity in a mouse melanoma model. Moreover, TILs overexpressing PGC-1α maintain higher mitochondrial activity and improved expansion when rechallenged in a tumor-free host. Altogether, our findings indicate that enforcing mitochondrial biogenesis promotes CD8 T cell memory formation, metabolic fitness, and antitumor immunity in vivo

Staphylococcus aureus bacteriuria as a prognosticator for outcome of Staphylococcus aureus bacteremia: a case-control study

<p>Abstract</p> <p>Background</p> <p>When <it>Staphylococcus aureus </it>is isolated in urine, it is thought to usually represent hematogenous spread. Because such spread might have special clinical significance, we evaluated predictors and outcomes of <it>S. aureus </it>bacteriuria among patients with <it>S. aureus </it>bacteremia.</p> <p>Methods</p> <p>A case-control study was performed at John H. Stroger Jr. Hospital of Cook County among adult inpatients during January 2002-December 2006. Cases and controls had positive and negative urine cultures, respectively, for <it>S. aureus</it>, within 72 hours of positive blood culture for <it>S. aureus</it>. Controls were sampled randomly in a 1:4 ratio. Univariate and multivariable logistic regression analyses were done.</p> <p>Results</p> <p>Overall, 59% of patients were African-American, 12% died, 56% of infections had community-onset infections, and 58% were infected with methicillin-susceptible <it>S. aureus </it>(MSSA). Among 61 cases and 247 controls, predictors of <it>S. aureus </it>bacteriuria on multivariate analysis were urological surgery (OR = 3.4, p = 0.06) and genitourinary infection (OR = 9.2, p = 0.002). Among patients who died, there were significantly more patients with bacteriuria than among patients who survived (39% vs. 17%; p = 0.002). In multiple Cox regression analysis, death risks in bacteremic patients were bacteriuria (hazard ratio 2.9, CI 1.4-5.9, p = 0.004), bladder catheter use (2.0, 1.0-4.0, p = 0.06), and Charlson score (1.1, 1.1-1.3, p = 0.02). Neither length of stay nor methicillin-resistant <it>Staphylococcus aureus </it>(MRSA) infection was a predictor of <it>S. aureus </it>bacteriuria or death.</p> <p>Conclusions</p> <p>Among patients with <it>S. aureus </it>bacteremia, those with <it>S. aureus </it>bacteriuria had 3-fold higher mortality than those without bacteriuria, even after adjustment for comorbidities. Bacteriuria may identify patients with more severe bacteremia, who are at risk of worse outcomes.</p

Temporal Regulation of Rapamycin on Memory CTL Programming by IL-12

Mammalian target of rapamycin (mTOR) is a master regulator of cell growth. Recent reports have defined its important role in memory cytotoxic T lymphocyte (CTL) differentiation in infections and memory programming. We report that rapamycin regulated memory CTL programming by IL-12 to a similar level in a wide range of concentrations, and the enhanced memory CTLs by rapamycin were functional and provided similar protection against Listeria Monocytogenes challenge compared to the control. In addition, rapamycin-experienced CTLs went through substantially enhanced proliferation after transfer into recipients. Furthermore, the regulatory function of rapamycin on CD62L expression in memory CTLs was mainly contributed by the presence of rapamycin in the first 24-hr of stimulation in vitro, whereas the effective window of rapamycin on the size of memory CTLs was determined between 24 to 72 hrs. In conclusion, rapamycin regulates IL-12-driven programming of CTLs to a similar level in a wide range of concentrations, and regulates the phenotype and the size of memory CTLs in different temporal windows

Global Activation of CD8+ Cytotoxic T Lymphocytes Correlates with an Impairment in Regulatory T Cells in Patients with Generalized Vitiligo

Melanocyte-specific CD8+ cytotoxic T lymphocytes (CTLs) play a pivotal role in vitiligo-induced depigmentation. Yet, the mechanisms underlying the high frequency of generalized autoimmune disorders associated with generalized vitiligo (GV) are unknown. We hypothesized that an imbalance between activated CD8+ CTLs and regulatory T cells (Tregs) exists in patients with GV . Assessment of the circulating CD8+ CTLs and Tregs by flow cytometric analysis revealed an obvious expansion of CD8+ CTLs and a concomitant decrease in Treg cells in GV patients. The percentages of skin infiltrating CD8+ CTLs and Tregs were evaluated by immunohistochemistry and revealed dramatically increased numbers of both CD8+ CTLs and Tregs in the perilesional skin of GV patients. However, peripheral Tregs were impaired in their ability to suppress the proliferation and cytolytic capacity of autologous CD8+ T cells, suggesting that a functional failure of Tregs and the hyper-activation of CD8+ CTLs may contribute to progressive GV. Our data indicate that reduced numbers and impaired function of natural Tregs fail to control the widespread activation of CD8+ CTLs, which leads to the destruction of melanocytes and contributes to the elevated frequency of various associated autoimmune diseases. This knowledge furthers our understanding of the mechanisms of immune tolerance that are impaired in GV patients and may aid in the future development of effective immunotherapy for GV patients