Bcl-xL regulates CD1d-mediated antigen presentation to NKT cells by altering CD1d trafficking through the endocytic pathway.

NKT cells are a unique subset of T cells that recognize glycolipid Ags presented in the context of CD1d molecules. NKT cells mount strong antitumor responses and are a major focus in developing effective cancer immunotherapy. It is known that CD1d molecules are constantly internalized from the cell surface, recycled through the endocytic compartments, and re-expressed on the cell surface. However, little is known about the regulation of CD1d-mediated Ag processing and presentation in B cell lymphoma. Prosurvival factors of the Bcl-2 family, such as Bcl-xL, are often upregulated in B cell lymphomas and are intimately linked to sphingolipid metabolism, as well as the endocytic compartments. We hypothesized that Bcl-xL can regulate CD1d-mediated Ag presentation to NKT cells. We found that overexpression or induction of Bcl-xL led to increased Ag presentation to NKT cells. Conversely, the inhibition or knockdown of Bcl-xL led to decreased NKT cell activation. Furthermore, knockdown of Bcl-xL resulted in the loss of CD1d trafficking to lysosome-associated membrane protein 1(+) compartments. Rab7, a late endosomal protein, was upregulated and CD1d molecules accumulated in the Rab7(+) late endosomal compartment. These results demonstrate that Bcl-xL regulates CD1d-mediated Ag processing and presentation to NKT cells by altering the late endosomal compartment and changing the intracellular localization of CD1d.

The natural tumorcide Manumycin-A targets protein phosphatase 1α and reduces hydrogen peroxide to induce lymphoma apoptosis.

Numerous compounds for treating human disease have been discovered in nature. Manumycin-A (Man-A) is a natural, well-tolerated microbial metabolite and a potent experimental tumoricide. We recently showed that Man-A stimulated reactive oxygen species (ROS) which were upstream of serine/threonine (Ser/Thr) dephosphorylation and caspase-dependent cleavage of MEK and Akt in lymphoma apoptosis. Conversely, activation-specific, Ser/Thr phosphorylation of MEK and Akt proteins was stable in Man-A-resistant tumors suggesting that stimulation of Ser/Thr PPase activity might be required for Man-A tumoricidal activity. Pre-treatment with Calyculin-A, an equipotent inhibitor of PP1 and PP2A, blocked all downstream effects of Man-A whereas, the PP2A-selective inhibitor, Okadaic acid did not, suggesting that PP1 and not PP2A played a role in Man-A action. Phosphorylation of PP1α on Thr320 inhibits its activity. Hence, we posited that if PP1α was important for Man-A action, then Man-A treatment should promote dephosphorylation of PP1α on Thr320. Indeed, T320 was only dephosphorylated in the tumors that underwent apoptosis. Lastly, stable over-expression of a constitutively active PP1α mimetic (PP1αT320A mutant), elevated basal ROS levels and enhanced Man-A-stimulated apoptosis. Taken together, we conclude that PP1α is an important proximal effector of Man-A mediated lymphoma apoptosis and that the mechanisms of Man-A action warrant further investigation.

Estimating the sex-specific effects of genes on facial attractiveness and sexual dimorphism.

Human facial attractiveness and facial sexual dimorphism (masculinity-femininity) are important facets of mate choice and are hypothesized to honestly advertise genetic quality. However, it is unclear whether genes influencing facial attractiveness and masculinity-femininity have similar, opposing, or independent effects across sex, and the heritability of these phenotypes is poorly characterized. To investigate these issues, we assessed facial attractiveness and facial masculinity-femininity in the largest genetically informative sample (n = 1,580 same- and opposite-sex twin pairs and siblings) to assess these questions to date. The heritability was ~0.50-0.70 for attractiveness and ~0.40-0.50 for facial masculinity-femininity, indicating that, despite ostensible selection on genes influencing these traits, substantial genetic variation persists in both. Importantly, we found evidence for intralocus sexual conflict, whereby alleles that increase masculinity in males have the same effect in females. Additionally, genetic influences on attractiveness were shared across the sexes, suggesting that attractive fathers tend to have attractive daughters and attractive mothers tend to have attractive sons.

A Multi-Institutional Description of Processes and Outcomes of Postbaccalaureate Research Education Programs in the Mid-Atlantic Region.

ABSTRACT: Outcome data from 6 National Institutes of Health-funded Postbaccalaureate Research Education Programs (PREPs) in the Mid-Atlantic region were combined to give a multi-institutional perspective on their scholars' characteristics and progress through biomedical research training. The institutions hosting these programs were Johns Hopkins University School of Medicine, the Medical University of South Carolina, the University of Maryland School of Medicine, the University of North Carolina at Chapel Hill, Virginia Commonwealth University, and Virginia Polytechnic Institute and State University. The authors summarize the institutional pathways, demographics, undergraduate institutions, and graduate institutions for a total of 384 PREP scholars who completed the programs by June 2021. A total of 228 (59.4%) of these PREP scholars identified as Black or African American, 116 (30.2%) as Hispanic or Latinx, and 269 (70.0%) as female. The authors found that 376 of 384 scholars (97.9%) who started PREP finished their program, 319 of 376 (84.8%) who finished PREP matriculated into PhD or MD/PhD programs, and 284 of 319 (89.0%) who matriculated have obtained their PhD or are successfully making progress toward their PhD.

Anti-MS4a4B treatment abrogates MS4a4B-mediated protection in T cells and ameliorates experimental autoimmune encephalomyelitis.

Recent data show that anti-CD20 therapy is effective for some autoimmune diseases, including multiple sclerosis (MS). However, the efficacy of anti-CD20 therapy for MS is largely limited because anti-CD20 antibodies target only B cells. In previous studies, we have investigated the function of MS4a4B, a novel CD20 homologue, in T cell proliferation. Here, we found that MS4a4B regulates not only T cell proliferation but also T cell apoptosis. Knockdown of MS4a4B by MS4a4B-siRNA or MS4a4B-shRNA-expressing vector promoted apoptosis in primary T cells and T32 cell line. In contrast, vector-driven over-expression of MS4a4B reduced apoptosis in EL-4 cells. Machinery analysis showed that MS4a4B-mediated T cell survival was associated with decreased activity of caspases 3, 8 and 9. Interestingly, binding of anti-MS4a4B antibodies to T cells induced activated T cells to undergo apoptosis. To test whether anti-MS4a4B antibody interferes with MS4a4B-mediated protection of T cells, we injected anti-MS4a4B antibodies into mice with experimental autoimmune encephalomyelitis (EAE). The results show that anti-MS4a4B treatment ameliorated the severity of EAE, accompanied by decreased Th1 and Th17 cell responses and reduced levels of pro-inflammatory cytokines in the central nervous system, suggesting that MS4a4B may serve as a target of antibody-based therapy for T cell-mediated diseases.

Insulin receptor substrate 1 expression enhances the sensitivity of 32D cells to chemotherapy-induced cell death.

The adapters IRS1 and IRS2 link growth factor receptors to downstream signaling pathways that regulate proliferation and survival. Both suppress factor-withdrawal-induced apoptosis and have been implicated in cancer progression. However, recent studies suggest IRS1 and IRS2 mediate differential functions in cancer pathogenesis. IRS1 promoted breast cancer proliferation, while IRS2 promoted metastasis. The role of IRS1 and IRS2 in controlling cell responses to chemotherapy is unknown. To determine the role of IRS1 and IRS2 in the sensitivity of cells to chemotherapy, we treated 32D cells lacking or expressing IRS proteins with various concentrations of chemotherapeutic agents. We found that expression of IRS1, in contrast to IRS2, enhanced the sensitivity of 32D cells to chemotherapy-induced apoptosis. When IRS2 was expressed with IRS1, the cells no longer showed enhanced sensitivity. Expression of IRS1 did not alter the expression of pro- and anti-apoptotic proteins; however, 32D-IRS1 cells expressed higher levels of Annexin A2. In 32D-IRS1 cells, IRS1 and Annexin A2 were both located in cytoplasmic and membrane fractions. We also found that IRS1 coprecipitated with Annexin A2, while IRS2 did not. Decreasing Annexin A2 levels reduced 32D-IRS1 cell sensitivity to chemotherapy. These results suggest IRS1 enhances sensitivity to chemotherapy in part through Annexin A2.

Adolescent peer choice and cigarette smoking: evidence of active gene-environment correlation?

Both peer groups and genetics have been associated with adolescent smoking behavior. Recently, Loehlin (Loehlin, J. C. (2010). Is there an active gene-environment correlation in adolescent drinking behavior? Behavior Genetics, 40, 447-451) reported that twin differences in alcohol use were associated with differences in the number of common friends. Twins with more common friends were more similar in drinking, but only for dizygotic pairs. Using the same sample as Loehlin's (the National Merit twins), we replicated all of these findings for a composite cigarette smoking measure and for smoking initiation, but not persistence. The pattern of results is most consistent with homophily, or the tendency to associate with individuals that are like oneself. If peer influence occurs in the presence of homophily, then active genotype-environment correlation will be induced.

Baltimore pediatric ocular trauma study: Health disparities and outcomes in pediatric and adolescent open globe trauma.

Purpose Children represent approximately one-third of patients with serious ocular injuries. Our study evaluates associations between race and socioeconomic status in presentation and outcomes of pediatric and adolescent traumatic open globe injuries. Methods We conducted a retrospective chart review of traumatic open globe injuries in pediatric and adolescent patients presenting to Johns Hopkins Hospital and University of Maryland Medical Center between 2006 and 2020. Variables assessed included age, gender, parent-identified race, median household income, mechanism of injury, initial and final visual acuity (VA), and length of follow-up. Results Eighty patients ranging from 4 months to 17.7 years (mean 9.3 years) presented with traumatic open globe injury. Identifications were 28 White (35%), 38 Black (48%), and 5 Hispanic (6%). Initial presenting and final VA, pediatric ocular trauma score (POTS), and length of follow-up did not differ significantly among race, gender, or income. Black patients had higher rates of blunt trauma (odds ratio (OR) 3.81; 95% confidence interval (CI) 0.95-15.24, p = 0.07), uveal prolapse (OR 3.58; 95% CI 1.03-12.43; p = 0.049), and enucleation (OR 10.55; 95% CI 1.26-88.31). Hispanic patients presented at a younger age of 2.8 years mean age vs. 9.9 years (p = 0.004) for others. Conclusion Visual outcomes following traumatic open globe injury were independent of race, gender, or income. However, blunt trauma, uveal prolapse, and enucleation rates were higher in Black patients, and ocular trauma occurred at a younger age in Hispanic patients.

Cognitive-behavioral group therapy versus group psychotherapy for social anxiety disorder among college students: a randomized controlled trial.

OBJECTIVE: In this randomized controlled trial, cognitive-behavioral group therapy (CBGT) for social anxiety disorder (SAD) was compared to group psychotherapy (GPT), a credible, structurally equivalent control condition that included only nonspecific factors of group treatment (such as group dynamics). METHODS: Participants were 45 college students at the University of Colorado with a primary diagnosis of SAD. Each treatment condition comprised eight group sessions lasting 2 hr each. Independent assessors (blind to treatment assignment) assessed participants at baseline and posttreatment with the Clinical Global Impression Scale (CGI) and the Liebowitz Social Anxiety Scale (LSAS). RESULTS: Both treatments were found to be equally credible. There were five noncompleters in the CBGT condition (21.7%) and only one in the GPT condition (4.3%). There were no statistically significant differences posttreatment (controlling for pretreatment scores) between the two treatment conditions, and both treatments were found to be efficacious. Effect sizes for CBGT were similar to earlier studies, and adherence ratings revealed excellent adherence. CONCLUSIONS: Treatment of SAD appears to be moving toward individual CBT, partly because of high attrition rates and underutilization of group dynamics in group CBT. However, group therapy has unique therapeutic ingredients, and it may be too early to give up on group treatment altogether. Discussion of these findings included future directions with this treatment modality, especially whether these two types of group treatment could be combined and whether such combination might serve to decrease attrition, enhance efficacy, and facilitate dissemination.

Targeting Natural Killer T Cells in Solid Malignancies.

Natural killer T (NKT) cells are a unique subset of lymphocytes that recognize lipid antigens in the context of the non-classical class I MHC molecule, CD1d, and serve as a link between the innate and adaptive immune system through their expeditious release of cytokines. Whereas NKT have well-established roles in mitigating a number of human diseases, herein, we focus on their role in cancer. NKT cells have been shown to directly and indirectly mediate anti-tumor immunity and manipulating their effector functions can have therapeutic significances in treatment of cancer. In this review, we highlight several therapeutic strategies that have been used to harness the effector functions of NKT cells to target different types of solid tumors. We also discuss several barriers to the successful utilization of NKT cells and summarize effective strategies being developed to harness the unique strengths of this potent population of T cells. Collectively, studies investigating the therapeutic potential of NKT cells serve not only to advance our understanding of this powerful immune cell subset, but also pave the way for future treatments focused on the modulation of NKT cell responses to enhance cancer immunotherapy.

B cells induce tolerance by presenting endogenous peptide-IgG on MHC class II molecules via an IFN-gamma-inducible lysosomal thiol reductase-dependent pathway.

We have previously demonstrated that splenic B cells, transduced with peptide-IgG fusion proteins, are efficient tolerogenic APCs in vivo. Specific hyporesponsiveness to epitopes encoded in the peptide-IgG fusion protein has been achieved to over one dozen Ags, and clinical efficacy has been established in animal models for several autoimmune diseases and hemophilia. Previous studies also demonstrated that tolerance in this system requires MHC class II expression by the transduced B cells. Yet, the mechanisms of this B cell tolerogenic processing pathway remain unclear. In this study, we show that MHC class II molecules on tolerogenic B cells present epitopes derived from endogenous, but not exogenous (secreted), peptide-IgG fusion protein. These class II epitopes from the IgG fusion protein are processed in lysosomes/endosomes in an IFN-gamma-inducible lysosomal thiol reductase-dependent manner. We suggest that the MHC class II presentation of endogenously produced fusion protein epitopes represents a novel mechanism for tolerance induced by peptide-IgG-transduced B cells. An understanding of this process might provide insights into central and peripheral tolerance induced by other professional and nonprofessional APCs.

Reactive oxygen species-dependent destruction of MEK and Akt in Manumycin stimulated death of lymphoid tumor and myeloma cell lines.

Manumycin-A (Man-A) is a farnesyltransferase inhibitor (FTI), which was originally identified as an effective tumoricide against several cancers, especially ones harboring constitutively active Ras. However, it is becoming apparent that Man-A can stimulate tumor death independently of FTases. Antioxidant treatment blocked Man-A-stimulated DNA damage and reversed Man-A-inhibited tumor growth. However, the precise molecular details of how these reactive oxygen species (ROS) influence cell signaling modules are poorly understood. We examined how ROS may modulate death and survival pathways in a panel of tumor cells. Man-A treatment resulted in a massive induction of superoxide anion (.O(2) (-)) only in Man-A-sensitive tumors. Within 1 hr, Man-A caused the ROS-dependent activation of caspases 9 and 3. In this time-frame, the Ras-Raf target, MEK, and the survival protein Akt were dephosphorylated in ROS-dependent fashions and then cleaved in ROS and caspase-dependent manners. Pretreatment with ROS scavengers blocked the adverse effects of Man-A, including the processing of caspases and the cleavage of MEK and Akt. These events were noted before any losses in Ras activity or changes in its maturation could be detected. Finally, transfection with cDNAs encoding the antioxidant enzymes catalase, superoxide dismutase and thioredoxin reductase inhibited superoxide induction and apoptosis. Together, our data suggest that the elimination of tumors by Man-A can be independent of the inhibiting of Ras. However, one universal feature observed is the generation of death-triggering intracellular oxidants that appear to directly participate in the select targeting of growth and survival proteins that then either augment or ensure tumor cell death.

Alterations in cellular metabolism modulate CD1d-mediated NKT-cell responses.

Natural killer T (NKT) cells play a critical role in the host's innate immune response. CD1d-mediated presentation of glycolipid antigens to NKT cells has been established; however, the mechanisms by which NKT cells recognize infected or cancerous cells remain unclear. 5(')-AMP activated protein kinase (AMPK) is a master regulator of lipogenic pathways. We hypothesized that activation of AMPK during infection and malignancy could alter the repertoire of antigens presented by CD1d and serve as a danger signal to NKT cells. In this study, we examined the effect of alterations in metabolism on CD1d-mediated antigen presentation to NKT cells and found that an infection with lymphocytic choriomeningitis virus rapidly increased CD1d-mediated antigen presentation. Hypoxia inducible factors (HIF) enhance T-cell effector functions during infection, therefore antigen presenting cells pretreated with pharmacological agents that inhibit glycolysis, induce HIF and activate AMPK were assessed for their ability to induce NKT-cell responses. Pretreatment with 2-deoxyglucose, cobalt chloride, AICAR and metformin significantly enhanced CD1d-mediated NKT-cell activation. In addition, NKT cells preferentially respond to malignant B cells and B-cell lymphomas express HIF-1α. These data suggest that targeting cellular metabolism may serve as a novel means of inducing innate immune responses.

Allometry and growth of eight tree taxa in United Kingdom woodlands.

As part of a project to develop predictive ecosystem models of United Kingdom woodlands we have collated data from two United Kingdom woodlands - Wytham Woods and Alice Holt. Here we present data from 582 individual trees of eight taxa in the form of summary variables relating to the allometric relationships between trunk diameter, height, crown height, crown radius and trunk radial growth rate to the tree's light environment and diameter at breast height. In addition the raw data files containing the variables from which the summary data were obtained. Large sample sizes with longitudinal data spanning 22 years make these datasets useful for future studies concerned with the way trees change in size and shape over their life-span.

Rapid detection of an ABT-737-sensitive primed for death state in cells using microplate-based respirometry.

Cells that exhibit an absolute dependence on the anti-apoptotic BCL-2 protein for survival are termed "primed for death" and are killed by the BCL-2 antagonist ABT-737. Many cancers exhibit a primed phenotype, including some that are resistant to conventional chemotherapy due to high BCL-2 expression. We show here that 1) stable BCL-2 overexpression alone can induce a primed for death state and 2) that an ABT-737-induced loss of functional cytochrome c from the electron transport chain causes a reduction in maximal respiration that is readily detectable by microplate-based respirometry. Stable BCL-2 overexpression sensitized non-tumorigenic MCF10A mammary epithelial cells to ABT-737-induced caspase-dependent apoptosis. Mitochondria within permeabilized BCL-2 overexpressing cells were selectively vulnerable to ABT-737-induced cytochrome c release compared to those from control-transfected cells, consistent with a primed state. ABT-737 treatment caused a dose-dependent impairment of maximal O(2) consumption in MCF10A BCL-2 overexpressing cells but not in control-transfected cells or in immortalized mouse embryonic fibroblasts lacking both BAX and BAK. This impairment was rescued by delivering exogenous cytochrome c to mitochondria via saponin-mediated plasma membrane permeabilization. An ABT-737-induced reduction in maximal O(2) consumption was also detectable in SP53, JeKo-1, and WEHI-231 B-cell lymphoma cell lines, with sensitivity correlating with BCL-2:MCL-1 ratio and with susceptibility (SP53 and JeKo-1) or resistance (WEHI-231) to ABT-737-induced apoptosis. Multiplexing respirometry assays to ELISA-based determination of cytochrome c redistribution confirmed that respiratory inhibition was associated with cytochrome c release. In summary, cell-based respiration assays were able to rapidly identify a primed for death state in cells with either artificially overexpressed or high endogenous BCL-2. Rapid detection of a primed for death state in individual cancers by "bioenergetics-based profiling" may eventually help identify the subset of patients with chemoresistant but primed tumors who can benefit from treatment that incorporates a BCL-2 antagonist.

TCR stimulation upregulates MS4a4B expression through induction of AP-1 transcription factor during T cell activation.

MS4a4B is a novel member of the membrane-spanning 4-domain family, subfamily A (MS4A) specifically expressed in mouse T cells. We have shown previously that expression of MS4a4B in T cells is upregulated upon T cell activation, suggesting that MS4a4B may play a functional role in regulation of T cell responses. However, little is known about the mechanisms that regulate MS4a4B gene expression. In this study, we explored the potential mechanism underlying TCR-stimulation-induced expression of MS4a4B by promoter analysis. We cloned 2495bp of 5'-flanking region upstream of the MS4a4B start code and inserted the DNA fragment into pGL4.20 reporter plasmid. To analyze promoter activity of the cloned DNA fragment, we transiently transfected EL4 thymoma cells and the T32 cell line with reporter plasmids. Expression of reporter gene was determined by dual-luciferase assay. Potential activator- and repressor-binding sites were analyzed by serial length of 5'-deletion. We have identified at least two potential activator binding regions and two potential repressor binding regions. The activator binding sites have been localized to two fragments, which are a 442-base pair region (region A) positioned from -1176 to -735, and a 119-base pair region (region B) positioned -188 to -70 respectively. MatInspector analysis showed that region A contains the consensus binding motif of the AP-1 family of transcription factors. Machinery analysis showed that nuclear proteins extracted from anti-CD3/anti-CD28-activated primary T cells specifically bind to the AP-1 binding element. In contrast, blockade by AP-1 inhibitor in culture decreased MS4a4B expression in T cells. Our data demonstrate that TCR-stimulation induces transactivation of AP-1 transcription factor, which subsequently binds to the MS4a4B promoter and upregulates expression of MS4a4B in activated T cells.

MS4a4B, a CD20 homologue in T cells, inhibits T cell propagation by modulation of cell cycle.

MS4a4B, a CD20 homologue in T cells, is a novel member of the MS4A gene family in mice. The MS4A family includes CD20, FcεRIß, HTm4 and at least 26 novel members that are characterized by their structural features: with four membrane-spanning domains, two extracellular domains and two cytoplasmic regions. CD20, FcεRIß and HTm4 have been found to function in B cells, mast cells and hematopoietic cells respectively. However, little is known about the function of MS4a4B in T cell regulation. We demonstrate here that MS4a4B negatively regulates mouse T cell proliferation. MS4a4B is highly expressed in primary T cells, natural killer cells (NK) and some T cell lines. But its expression in all malignant T cells, including thymoma and T hybridoma tested, was silenced. Interestingly, its expression was regulated during T cell activation. Viral vector-driven overexpression of MS4a4B in primary T cells and EL4 thymoma cells reduced cell proliferation. In contrast, knockdown of MS4a4B accelerated T cell proliferation. Cell cycle analysis showed that MS4a4B regulated T cell proliferation by inhibiting entry of the cells into S-G2/M phase. MS4a4B-mediated inhibition of cell cycle was correlated with upregulation of Cdk inhibitory proteins and decreased levels of Cdk2 activity, subsequently leading to inhibition of cell cycle progression. Our data indicate that MS4a4B negatively regulates T cell proliferation. MS4a4B, therefore, may serve as a modulator in the negative-feedback regulatory loop of activated T cells.