Trans-vaccenic acid reprograms CD8+ T cells and anti-tumour immunity.

Diet-derived nutrients are inextricably linked to human physiology by providing energy and biosynthetic building blocks and by functioning as regulatory molecules. However, the mechanisms by which circulating nutrients in the human body influence specific physiological processes remain largely unknown. Here we use a blood nutrient compound library-based screening approach to demonstrate that dietary trans-vaccenic acid (TVA) directly promotes effector CD8+ T cell function and anti-tumour immunity in vivo. TVA is the predominant form of trans-fatty acids enriched in human milk, but the human body cannot produce TVA endogenously1. Circulating TVA in humans is mainly from ruminant-derived foods including beef, lamb and dairy products such as milk and butter2,3, but only around 19% or 12% of dietary TVA is converted to rumenic acid by humans or mice, respectively4,5. Mechanistically, TVA inactivates the cell-surface receptor GPR43, an immunomodulatory G protein-coupled receptor activated by its short-chain fatty acid ligands6-8. TVA thus antagonizes the short-chain fatty acid agonists of GPR43, leading to activation of the cAMP-PKA-CREB axis for enhanced CD8+ T cell function. These findings reveal that diet-derived TVA represents a mechanism for host-extrinsic reprogramming of CD8+ T cells as opposed to the intrahost gut microbiota-derived short-chain fatty acids. TVA thus has translational potential for the treatment of tumours.

Tcf-1 protects anti-tumor TCR-engineered CD8+ T-cells from GzmB mediated self-destruction.

BACKGROUND: T-cell longevity is undermined by antigen-driven differentiation programs that render cells prone to attrition through several mechanisms. CD8 + T cells that express the Tcf-1 transcription factor have undergone limited differentiation and exhibit stem-cell-like replenishment functions that facilitate persistence. We engineered human CD8 + T cells to constitutively express Tcf-1 and a TCR specific for the NY-ESO-1 cancer-associated antigen. Co-engineered cells were assessed for their potential for adoptive cellular immunotherapy. METHODS: Tcf-1 mRNA encoding TCF-1B and TCF-1E isoforms, along with GzmB expression were assessed in CD62L + CD57 -, CD62L - CD57 -, and CD62L - CD57 + CD8 + T cells derived from normal donor lymphocytes. The impact of stable Tcf-1B expression on CD8 + T-cell phenotype, anti-tumor activity, and cell-cycle activity was assessed in vitro and in an in vivo tumor xenograft model. RESULTS: TCF-1B and TCF-1E were dynamically regulated during self-renewal, with progeny of recently activated naïve T cells more enriched for TCF-1B mRNA. Constitutive TCF-1B expression improved the survival of TCR-engineered CD8 + T cells upon engagement with tumor cells. Tcf-1B prohibited the acquisition of a GzmB High state, and protected T cells from apoptosis associated with elicitation of effector function, and promoted stem cell-like characteristics. CONCLUSIONS: Tcf-1 protects TCR-engineered CD8 + T cells from activation induced cell death by restricting GzmB expression. Our study presents constitutive Tcf-1B expression as a potential means to impart therapeutic T cells with attributes of persistence for durable anti-tumor activity.

Multisite Study of Evoked Potentials in Rett Syndrome.

OBJECTIVE: The aim of the current study was to evaluate the utility of evoked potentials as a biomarker of cortical function in Rett syndrome (RTT). As a number of disease-modifying therapeutics are currently under development, there is a pressing need for biomarkers to objectively and precisely assess the effectiveness of these treatments. METHOD: Yearly visual evoked potentials (VEPs) and auditory evoked potentials (AEPs) were acquired from individuals with RTT, aged 2 to 37 years, and control participants across 5 sites as part of the Rett Syndrome and Related Disorders Natural History Study. Baseline and year 1 data, when available, were analyzed and the repeatability of the results was tested. Two syndrome-specific measures from the Natural History Study were used for evaluating the clinical relevance of the VEP and AEP parameters. RESULTS: At the baseline study, group level comparisons revealed reduced VEP and AEP amplitude in RTT compared to control participants. Further analyses within the RTT group indicated that this reduction was associated with RTT-related symptoms, with greater severity associated with lower VEP and AEP amplitude. In participants with RTT, VEP and AEP amplitude was also negatively associated with age. Year 1 follow-up data analyses yielded similar findings and evidence of repeatability of EPs at the individual level. INTERPRETATION: The present findings indicate the promise of evoked potentials (EPs) as an objective measure of disease severity in individuals with RTT. Our multisite approach demonstrates potential research and clinical applications to provide unbiased assessment of disease staging, prognosis, and response to therapy. ANN NEUROL 2021;89:790-802.

Stimulation of an anti-tumor immune response with "chromatin-damaging" therapy.

Curaxins are small molecules that bind genomic DNA and interfere with DNA-histone interactions leading to the loss of histones and decondensation of chromatin. We named this phenomenon 'chromatin damage'. Curaxins demonstrated anti-cancer activity in multiple pre-clinical tumor models. Here, we present data which reveals, for the first time, a role for the immune system in the anti-cancer effects of curaxins. Using the lead curaxin, CBL0137, we observed elevated expression of several group of genes in CBL0137-treated tumor cells including interferon sensitive genes, MHC molecules, some embryo-specific antigens suggesting that CBL0137 increases tumor cell immunogenicity and improves recognition of tumor cells by the immune system. In support of this, we found that the anti-tumor activity of CBL0137 was reduced in immune deficient SCID mice when compared to immune competent mice. Anti-tumor activity of CBL0137 was abrogated in CD8+ T cell depleted mice but only partially lost when natural killer or CD4+ T cells were depleted. Further support for a key role for the immune system in the anti-tumor activity of CBL0137 is evidenced by an increased antigen-specific effector CD8+ T cell and NK cell response, and an increased ratio of effector T cells to Tregs in the tumor and spleen. CBL0137 also elevated the number of CXCR3-expressing CTLs in the tumor and the level of interferon-γ-inducible protein 10 (IP-10) in serum, suggesting IP-10/CXCR3 controls CBL0137-elicited recruitment of effector CTLs to tumors. Our collective data underscores a previously unrecognized role for both innate and adaptive immunity in the anti-tumor activity of curaxins.

Native non-prototypicality in vowel perception induces prominent neuromagnetic mismatch intensities in non-native speakers: a pilot study.

Neural mismatch response resulting from the difference between prediction and observation is related to change detection and discrimination. Robust neuromagnetic brain activity of auditory mismatch-related perception occurs in response to non-prototypical vowels in across-category contrasts for first-language speakers. However, whether this non-prototypicality effect applies to within-category vowel perception remains to be elucidated. Here, healthy Japanese adults (n = 7) were subjected to magnetoencephalography (MEG) while watching a silent movie, and passively listened to synthesized English vowels /i/. We observed the source-level mismatch effect to the mid-high near-front vowel deviant [ɪ] with the most non-prototypical, unspecified feature in the participants' native language system. The mismatch effect recruited the left posterior superior temporal sulcus with a peak latency of 225 ms post-stimulus onset. We further studied whether a longer F1 distance between vowel pairs would increase mismatch-activated intensities, however, we did not observe neuromagnetic changes when the prototypical anchor standard [i] was compared with three non-prototypical deviants differing in first resonance frequency (F1) values. Our results indicate that an F1 increase in within-category upper front vowel perception is a strong activator of mismatch responses measured by source-level activated intensities for non-native listeners.

Head errors of syntactic dependency increase neuromagnetic mismatch intensities.

Mismatch-related brain activation in healthy individuals is an important area of neural investigation. Previously, we evaluated sentence-level syntactic dependencies, composed of a head and a dependent between two syntactically related words in head-initial English structures. We demonstrated that prominent mismatch effects were induced by within-category dependent errors when semantic interpretation was preserved. However, the following issues were not addressed: (1) whether head errors of syntactic dependency in head-final structures would elicit large mismatch field (MMF) intensities, and (2) whether an MMF effect of syntactic errors would be seen in the left superior temporal cortex alone. In this study, auditory MMFs were obtained by magnetocephalography (MEG) from healthy Japanese adults (n = 8) who were subjected to a passive auditory oddball paradigm with syntactically legal or illegal utterances and single words in Japanese. The results demonstrate that the source waveforms had significantly higher MMF cortical activation in response to the head error, which involved altered polarity of the predicate. This resulted in a syntactically incorrect and semantically incomprehensible expression, when compared to the syntactically correct expression and the non-structural lexical item. This mismatch effect, with a peak latency of 164 ms, was confined to the anterior region of the left superior temporal cortex. The current results clearly indicate that the representation of syntactic dependency is stored in long-term memory and tends to be activated in automatic auditory processing.

An Immunosuppressive Effect of Melanoma-derived Exosomes on NY-ESO-1 Antigen-specific Human CD8+ T Cells is Dependent on IL-10 and Independent of BRAFV600E Mutation in Melanoma Cell Lines.

Exosomes, including human melanoma-derived exosomes (HMEX), are known to suppress the function of immune effector cells, which for HMEX has been associated with the surface presence of the immune checkpoint ligand PD-L1. This study investigated the relationship between the BRAF mutational status of melanoma cells and the inhibition of secreted HMEX exosomes on antigen-specific human T cells. Exosomes were isolated from two melanoma cell lines, 2183-Her4 and 888-mel, which are genetically wild-type BRAFWT and BRAFV600E, respectively. HMEX were isolated using a modified, size-exclusion chromatography (SEC) method shown to reduce co-isolation of non-exosome-associated cytokines compared to ultracentrifugation isolation. The immunoinhibitory effect of the exosomes was tested in vitro on patient-derived NY-ESO-1-specific CD8+ T cells challenged with NY-ESO-1 antigen. HMEX from both cell lines inhibited the immune response of antigen-specific T cells comparably, as evidenced by the reduction of IFN-γ and TNF-α in NY-ESO-1 tetramer-positive cells. This inhibition could be partially reversed by the presence of anti-PD-L1 and anti-IL-10 antibodies. IL-10 has been demonstrated to be a critical pathway for sustaining enhanced tumorigenesis in BRAFV600E mutant cells compared to BRAFWT melanoma cells. Thus, we demonstrate that HMEX inhibit antigen-specific T cell responses independent of the BRAF mutational status of the parent cells. In addition, PD-L1 and IL-10 contribute to the HMEX-mediated immunosuppression of antigen-specific human T cells. The inhibitory capacity of exosomes should be taken into consideration when developing therapies that are reliant upon the potency of customized, antigen-specific effector T cells.

Sublethal Radiation Affects Antigen Processing and Presentation Genes to Enhance Immunogenicity of Cancer Cells.

While immunotherapy in cancer is designed to stimulate effector T cell response, tumor-associated antigens have to be presented on malignant cells at a sufficient level for recognition of cancer by T cells. Recent studies suggest that radiotherapy enhances the anti-cancer immune response and also improves the efficacy of immunotherapy. To understand the molecular basis of such observations, we examined the effect of ionizing X-rays on tumor antigens and their presentation in a set of nine human cell lines representing cancers of the esophagus, lung, and head and neck. A single dose of 7.5 or 15 Gy radiation enhanced the New York esophageal squamous cell carcinoma 1 (NY-ESO-1) tumor-antigen-mediated recognition of cancer cells by NY-ESO-1-specific CD8+ T cells. Irradiation led to significant enlargement of live cells after four days, and microscopy and flow cytometry revealed multinucleation and polyploidy in the cells because of dysregulated mitosis, which was also revealed in RNA-sequencing-based transcriptome profiles of cells. Transcriptome analyses also showed that while radiation had no universal effect on genes encoding tumor antigens, it upregulated the expression of numerous genes involved in antigen processing and presentation pathways in all cell lines. This effect may explain the immunostimulatory role of cancer radiotherapy.

Abnormal Auditory Mismatch Fields in Children and Adolescents with 47,XYY Syndrome.

47,XYY syndrome (XYY) is one of the common forms of sex chromosome aneuploidy in males. XYY males tend to have tall stature, early speech, motor delays, social and behavioral challenges, and a high rate of language impairment. Recent studies indicate that 20-40% of males with XYY meet diagnostic criteria for autism spectrum disorder (ASD; the rate in the general population is 1-2%). Although many studies have examined the neural correlates of language impairment in ASD, few similar studies have been conducted on individuals with XYY. Studies using magnetoencephalography (MEG) in idiopathic ASD (ASD-I) have demonstrated delayed neurophysiological responses to changes in the auditory stream, revealed in the mismatch negativity or its magnetic counterpart, the mismatch field (MMF). This study investigated whether similar findings are observed in XYY-associated ASD and whether delayed processing is also present in individuals with XYY without ASD. MEG measured MMFs arising from the left and the right superior temporal gyrus during an auditory oddball paradigm with vowel stimuli (/a/ and /u/) in children/adolescents with XYY both with and without a diagnosis of ASD, as well as in those with ASD-I and in typically developing controls (TD). Ninety male participants (6-17 years old) were included in the final analyses (TD, n = 38, 11.50 ± 2.88 years; ASD-I, n = 21, 13.83 ± 3.25 years; XYY without ASD, n = 15, 12.65 ± 3.91 years; XYY with ASD, n = 16, 12.62 ± 3.19 years). The groups did not differ significantly in age (p > 0.05). There was a main effect of group on MMF latency (p < 0.001). Delayed MMF latencies were found in participants with XYY both with and without an ASD diagnosis, as well as in the ASD-I group compared to the TD group (ps < 0.001). Furthermore, participants with XYY (with and without ASD) showed a longer MMF latency than the ASD-I group (ps < 0.001). There was, however, no significant difference in MMF latency between individuals with XYY with ASD and those with XYY without ASD. Delayed MMF latencies were associated with severity of language impairment. Our findings suggest that auditory MMF latency delays are pronounced in this specific Y chromosome aneuploidy disorder, both with and without an ASD diagnosis, and thus may implicate the genes of the Y chromosome in mediating atypical MMF activity.

Delayed Auditory Evoked Responses in Autism Spectrum Disorder across the Life Span.

The M50 and M100 auditory evoked responses reflect early auditory processes in the primary/secondary auditory cortex. Although previous M50 and M100 studies have been conducted on individuals with autism spectrum disorder (ASD) and indicate disruption of encoding simple sensory information, analogous investigations of the neural correlates of auditory processing through development from children into adults are very limited. Magnetoencephalography was used to record signals arising from the left and right superior temporal gyrus during auditory presentation of tones to children/adolescents and adults with ASD as well as typically developing (TD) controls. One hundred and thirty-two participants (aged 6-42 years) were included into the final analyses (children/adolescents: TD, n = 36, 9.21 ± 1.6 years; ASD, n = 58, 10.07 ± 2.38 years; adults: TD, n = 19, 26.97 ± 1.29 years; ASD, n = 19, 23.80 ± 6.26 years). There were main effects of group on M50 and M100 latency (p < 0.001) over hemisphere and frequency. Delayed M50 and M100 latencies were found in participants with ASD compared to the TD group, and earlier M50 and M100 latencies were associated with increased age. Furthermore, there was a statistically significant association between language ability and both M50 and M100 latencies. Importantly, differences in M50 and M100 latencies between TD and ASD cohorts, often reported in children, persisted into adulthood, with no evidence supporting latency convergence.

Abnormal cortical activation during an auditory word comprehension task in benign childhood epilepsy with centrotemporal spikes: A magnetoencephalographic study.

OBJECTIVE: Benign childhood epilepsy with centrotemporal spikes (BECTS), also known as rolandic epilepsy, has recently been reported to be associated with variable degrees of cognitive dysfunction. Many studies reported poor language ability in children with BECTS compared with healthy control children. To elucidate the harmful effects of BECTS on language cognition, we studied the magnetoencephalographic activity elicited by an auditory language comprehension task. METHODS: The participants (N = 20) included 10 children diagnosed with BECTS (aged 10.8 ±â€¯2.8 years) and 10 age-matched healthy children (control) (aged 10.6 ±â€¯1.6 years). Cognitive function was assessed using general intellectual function and language ability. In patients with BECTS, we reviewed the clinical course and electroencephalogram (EEG) findings. We recorded the cortical responses elicited by an auditory language comprehension task using magnetoencephalography (MEG). We compared those results between groups and analyzed the correlation with cognitive scores and frequency of spikes. RESULTS: The full-scale intelligence quotient (FSIQ) by the Wechsler Intelligence Scale for Children-4th edition was significantly reduced in the group with BECTS (96.4 ±â€¯12.3) compared with the control group (110.0 ±â€¯7.4). In half of the group with BECTS, the auditory comprehension score fell below the age-standard level. In the group with BECTS, the cortical activation during the task showed reduced intensity in language-associated areas such as the bilateral primary auditory cortex, left superior and mid-temporal areas, and inferior frontal area compared with those in the control group. In addition, the cortical activation in the left superior temporal area was negatively correlated with spike frequency and positively correlated with FSIQ in the group with BECTS. Conversely, the right inferior frontal and mid-temporal areas had increased the activations in the group with BECTS. From the time frequency analysis, low gamma band event-related desynchronization was reduced in the group with BECTS. CONCLUSION: Epileptic spikes negatively influenced responsiveness to the auditory language comprehension task in the language-associated cortices. These findings suggest that epileptic spikes could have a negative impact on the functional activity in rolandic areas and become a reason to change the functional development of the language network.

NY-ESO-1 expression predicts an aggressive phenotype of ovarian cancer.

OBJECTIVES: NY-ESO-1 is a cancer testis antigen and a promising target for immunotherapy. The purpose of this study was to determine the expression frequency, immunogenicity, and clinical impact of NY-ESO-1 in ovarian cancer. METHODS: Immunohistochemistry (IHC), reverse-transcription polymerase chain reaction (RT-PCR), and quantitative-PCR (qRT-PCR) were utilized in an ovarian cancer (including Fallopian tube and primary peritoneal cancers) patient cohort; humoral responses against NY-ESO-1 were determined by ELISA. Clinicopathologic outcomes including progression-free (PFS) and overall (OS) survival were evaluated based on NY-ESO-1 expression. Cohen's kappa (κ) tested agreement between expression tests. RESULTS: NY-ESO-1 expression was detected by any method in 40.7% of 1002 patients' tumors (NY-ESO-1+) and baseline humoral response was identified in 19.0% of 689 tested patients. NY-ESO-1+ patients were older (p<0.001), higher stage (85% stage III/IV vs. 76.4%, p=0.015), less likely to have a complete response to initial therapy (53.9% vs. 68.9%, p=0.002), had more serous histotype (74.5% vs. 66.9%, p=0.011), and had more grade 3 tumors (83.7% vs. 70.8%, p<0.001). There was a trend towards shorter PFS (22.2 vs. 25.0months, p=0.07) and significantly shorter OS (42.9 vs. 50.0months, p=0.003) among NY-ESO-1+ patients. A subset analysis of NY-ESO-1+ patients that received immunotherapy demonstrated improved OS by >2years (52.6 vs. 27.2months, p<0.001). CONCLUSIONS: This study is the first demonstration of an association between NY-ESO-1 expression and an aggressive cancer phenotype. The relatively high expression frequency of NY-ESO-1 in ovarian cancer patients coupled with the poor clinical outcomes in NY-ESO-1+ patients reveals an underappreciated need for targeted therapy against this antigen. In support, our study reveals that NY-ESO-1+ patients enrolled on immunotherapy trials targeting the antigen exhibited an improvement in OS.

HLA superfamily assignment is a predictor of immune response to cancer testis antigens and survival in ovarian cancer.

OBJECTIVES: To characterize the association between major histocompatibility complex (MHC) types and spontaneous antibody development to the cancer testis (CT) antigen NY-ESO-1. METHODS: Tumor expression of NY-ESO-1 and serum antibodies to NY-ESO-1 were characterized in addition to human leukocyte antigen (HLA) type for patients with epithelial ovarian cancer. HLA types were assigned to structure-based superfamilies and statistical associations were examined. HLA types were compared to existing reference libraries of HLA frequencies in a European-Caucasian American population. RESULTS: Out of 126 patients identified, 81% were expression positive and 48% had spontaneous antibody responses to NY-ESO-1. There was an association between HLA-B superfamily and seropositivity among patients with tumors expressing NY-ESO-1 (p<0.001). The differences in HLA-B superfamily assignment were driven by HLA-B44. Among all patients, the B27 superfamily was over-represented compared with the general population (p<0.001). CONCLUSIONS: HLA type appears to be associated with spontaneous anti-CT antigen antibodies, as well as with the overall risk of ovarian cancer.

Efficacy of vaccination with recombinant vaccinia and fowlpox vectors expressing NY-ESO-1 antigen in ovarian cancer and melanoma patients.

Recombinant poxviruses (vaccinia and fowlpox) expressing tumor-associated antigens are currently being evaluated in clinical trials as cancer vaccines to induce tumor-specific immune responses that will improve clinical outcome. To test whether a diversified prime and boost regimen targeting NY-ESO-1 will result in clinical benefit, we conducted two parallel phase II clinical trials of recombinant vaccinia-NY-ESO-1 (rV-NY-ESO-1), followed by booster vaccinations with recombinant fowlpox-NY-ESO-1 (rF-NY-ESO-1) in 25 melanoma and 22 epithelial ovarian cancer (EOC) patients with advanced disease who were at high risk for recurrence/progression. Integrated NY-ESO-1-specific antibody and CD4(+) and CD8(+) T cells were induced in a high proportion of melanoma and EOC patients. In melanoma patients, objective response rate [complete and partial response (CR+PR)] was 14%, mixed response was 5%, and disease stabilization was 52%, amounting to a clinical benefit rate (CBR) of 72% in melanoma patients. The median PFS in the melanoma patients was 9 mo (range, 0-84 mo) and the median OS was 48 mo (range, 3-106 mo). In EOC patients, the median PFS was 21 mo (95% CI, 16-29 mo), and median OS was 48 mo (CI, not estimable). CD8(+) T cells derived from vaccinated patients were shown to lyse NY-ESO-1-expressing tumor targets. These data provide preliminary evidence of clinically meaningful benefit for diversified prime and boost recombinant pox-viral-based vaccines in melanoma and ovarian cancer and support further evaluation of this approach in these patient populations.

Heat shock protein 90-mediated peptide-selective presentation of cytosolic tumor antigen for direct recognition of tumors by CD4(+) T cells.

Tumor Ag-specific CD4(+) T cells play important functions in tumor immunosurveillance, and in certain cases they can directly recognize HLA class II-expressing tumor cells. However, the underlying mechanism of intracellular Ag presentation to CD4(+) T cells by tumor cells has not yet been well characterized. We analyzed two naturally occurring human CD4(+) T cell lines specific for different peptides from cytosolic tumor Ag NY-ESO-1. Whereas both lines had the same HLA restriction and a similar ability to recognize exogenous NY-ESO-1 protein, only one CD4(+) T cell line recognized NY-ESO-1(+) HLA class II-expressing melanoma cells. Modulation of Ag processing in melanoma cells using specific molecular inhibitors and small interfering RNA revealed a previously undescribed peptide-selective Ag-presentation pathway by HLA class II(+) melanoma cells. The presentation required both proteasome and endosomal protease-dependent processing mechanisms, as well as cytosolic heat shock protein 90-mediated chaperoning. Such tumor-specific pathway of endogenous HLA class II Ag presentation is expected to play an important role in immunosurveillance or immunosuppression mediated by various subsets of CD4(+) T cells at the tumor local site. Furthermore, targeted activation of tumor-recognizing CD4(+) T cells by vaccination or adoptive transfer could be a suitable strategy for enhancing the efficacy of tumor immunotherapy.

NK Receptor Signaling Lowers TCR Activation Threshold, Enhancing Selective Recognition of Cancer Cells by TAA-Specific CTLs.

Cytotoxic CD8+ T lymphocyte (CTL) recognition of non-mutated tumor-associated antigens (TAA), present on cancer cells and also in healthy tissues, is an important element of cancer immunity, but the mechanism of its selectivity for cancer cells and opportunities for its enhancement remain elusive. In this study, we found that CTL expression of the NK receptors (NKR) DNAM1 and NKG2D was associated with the effector status of CD8+ tumor-infiltrating lymphocytes and long-term survival of patients with melanoma. Using MART1 and NY-ESO-1 as model TAAs, we demonstrated that DNAM1 and NKG2D regulate T-cell receptor (TCR) functional avidity and set the threshold for TCR activation of human TAA-specific CTLs. Superior co-stimulatory effects of DNAM1 over CD28 involved enhanced TCR signaling, CTL killer function, and polyfunctionality. Double transduction of human CTLs with TAA-specific TCR and NKRs resulted in strongly enhanced antigen sensitivity, without a reduction in antigen specificity and selectivity of killer function. In addition, the elevation of NKR ligand expression on cancer cells due to chemotherapy also increased CTL recognition of cancer cells expressing low levels of TAAs. Our data help explain the ability of self-antigens to mediate tumor rejection in the absence of autoimmunity and support the development of dual-targeting adoptive T-cell therapies that use NKRs to enhance the potency and selectivity of recognition of TAA-expressing cancer cells.

A harmonized approach to intracellular cytokine staining gating: Results from an international multiconsortia proficiency panel conducted by the Cancer Immunotherapy Consortium (CIC/CRI).

Previous results from two proficiency panels of intracellular cytokine staining (ICS) from the Cancer Immunotherapy Consortium and panels from the National Institute of Allergy and Infectious Disease and the Association for Cancer Immunotherapy highlight the variability across laboratories in reported % CD8+ or % CD4+ cytokine-positive cells. One of the main causes of interassay variability in flow cytometry-based assays is due to differences in gating strategies between laboratories, which may prohibit the generation of robust results within single centers and across institutions. To study how gating strategies affect the variation in reported results, a gating panel was organized where all participants analyzed the same set of Flow Cytometry Standard (FCS) files from a four-color ICS assay using their own gating protocol (Phase I) and a gating protocol drafted by consensus from the organizers of the panel (Phase II). Focusing on analysis removed donor, assay, and instrument variation, enabling us to quantify the variability caused by gating alone. One hundred ten participating laboratories applied 110 different gating approaches. This led to high variability in the reported percentage of cytokine-positive cells and consequently in response detection in Phase I. However, variability was dramatically reduced when all laboratories used the same gating strategy (Phase II). Proximity of the cytokine gate to the negative population most impacted true-positive and false-positive response detection. Recommendations are provided for the (1) placement of the cytokine-positive gate, (2) identification of CD4+ CD8+ double-positive T cells, (3) placement of lymphocyte gate, (4) inclusion of dim cells, (5) gate uniformity, and 6) proper adjustment of the biexponential scaling.

Altered microstructural connectivity of the superior cerebellar peduncle is related to motor dysfunction in children with autistic spectrum disorders.

Many studies have reported motor impairments in autistic spectrum disorders (ASD). However, the brain mechanism underlying motor impairment in ASD remains unclear. Recent neuroimaging studies have suggested that underconnectivity between the cerebellum and other brain regions contributes to the features of ASD. In this study, we investigated the microstructural integrity of the cerebellar pathways, including the superior, middle, and inferior cerebellar peduncles, of children with and without ASD by using diffusion tensor imaging (DTI) tractography to determine whether the microstructural integrity of the cerebellar pathways is related to motor function in children with ASD. Thirteen children with ASD and 11 age-, gender-, handedness-, and IQ-matched typically developing (TD) controls were enrolled in this study. DTI outcome measurements, such as fractional anisotropy (FA), axial diffusivity (AD), and radial diffusivity (RD), for the cerebellar pathways were calculated. The Movement Assessment Battery for Children 2 (M-ABC 2) was used for assessing motor functions. There were no significant differences between the two groups in RD. However, compared to the TD subjects, patients with ASD had a significantly lower FA in the right superior cerebellar peduncle and lower AD in the left superior cerebellar peduncle, in addition to a significantly lower score in ball skills and the total test score of M-ABC 2. There was a significant positive correlation between the total test score of M-ABC 2 and FA in the right superior cerebellar peduncle in the ASD group. These findings suggest that the altered microstructural integrity of the superior cerebellar peduncle may be related to motor impairment in ASD.

Antibody-targeted NY-ESO-1 to mannose receptor or DEC-205 in vitro elicits dual human CD8+ and CD4+ T cell responses with broad antigen specificity.

Immunization of cancer patients with vaccines containing full-length tumor Ags aims to elicit specific Abs and both CD4(+) and CD8(+) T cells. Vaccination with protein Ags, however, often elicits only CD4(+) T cell responses without inducing Ag-specific CD8(+) T cells, as exogenous protein is primarily presented to CD4(+) T cells. Recent data revealed that Ab-mediated targeting of protein Ags to cell surface receptors on dendritic cells could enhance the induction of both CD4(+) and CD8(+) T cells. We investigated in this study if these observations were applicable to NY-ESO-1, a cancer-testis Ag widely used in clinical cancer vaccine trials. We generated two novel targeting proteins consisting of the full-length NY-ESO-1 fused to the C terminus of two human mAbs against the human mannose receptor and DEC-205, both internalizing molecules expressed on APC. These targeting proteins were evaluated for their ability to activate NY-ESO-1-specific human CD4(+) and CD8(+) T cells in vitro. Both targeted NY-ESO-1 proteins rapidly bound to their respective targets on APC. Whereas nontargeted and Ab-targeted NY-ESO-1 proteins similarly activated CD4(+) T cells, cross-presentation to CD8(+) T cells was only efficiently induced by targeted NY-ESO-1. In addition, both mannose receptor and DEC-205 targeting elicited specific CD4(+) and CD8(+) T cells from PBLs of cancer patients. Receptor-specific delivery of NY-ESO-1 to APC appears to be a promising vaccination strategy to efficiently generate integrated and broad Ag-specific immune responses against NY-ESO-1 in cancer patients.