Citations

Abstract TCR mediated antigen recognition results in a signaling cascade leading to T cell activation. Antigen binding to its cognate TCR drives receptor aggregation and conformational changes that trigger signaling from the receptor. Conformational change in the CD3 complex (CD3Δc) is required for robust signaling and productive T cell responses. In contrast, weakly recognized antigens do not induce CD3Δc and the resulting T cell response is poor. Thus, T cell responses to weak antigens may be improved by induction of CD3Δc. We previously described an anti-mouse CD3ɛ monovalent Fab (Mono-7D6-Fab) that induced CD3Δc and synergized with weak antigens to enhance the T cell response, a process we termed co-potentiation. However, Mono-7D6-Fab did not induce classical T cell signaling in the absence of antigenic ligation. Mono-7D6-Fab effectively co-potentiated T cells against murine B16-F10 melanoma cells and reduced tumor burden in vivo in a T cell dependent manner. We exploited a model of human cytomegalovirus (CMV) to generate proof of concept that co-potentiation may enhance T cell antigen recognition in humans. Anti-human CD3ɛ monovalent Fab (Mono-OKT3-Fab) improved the CD8 T cell response to the CMV pp65495–503 peptide as measured by increased T cell proliferation, cytokine production, and the ability to kill target cells. Co-potentiation was dependent on antigen specificity, as 1) the effects were dampened when antigen recognition was impaired and 2) sequencing of the TCRs revealed a restricted number of expanded TCR clones. Our results suggest that Mono-OKT3-Fab may have therapeutic clinical applications to achieve a more robust antigen-specific T cell response in the context of diverse human disease, from cancer to chronic infection.

Abstract Signaling networks and the protein interactions that define them are of great interest, but techniques to assess these networks in unmanipulated primary human cells are limited. We have developed and applied a novel technological and analytical platform to measure proteins in shared complexes detected by exposed surface epitopes (PiSCES). Immunoprecipitation reactions are performed in multiplex using microsphere beads to measure 20+ proteins and 210+ distinct pair-wise protein associations that are important for T cell antigen receptor signaling. Jurkat T cells stimulated with superantigen generated an extensive PiSCES signature using this method and provided a dataset from which we optimized an analytical package. The system is currently being utilized to address the hypothesis that T cells isolated from skin lesions of autoimmune alopecia areta (AA) patients will have a unique PiSCES signature compared to those isolated from non-AA donors. To investigate this, we have isolated non-engineered primary cells from AA patient skin lesions and healthy controls, and have begun to identify differences in their PiSCES signatures, either directly after isolation or after stimulation with anti-CD3 and anti-CD28. We conclude that this novel PiSCES approach is capable of generating protein-protein interaction signatures that provide a unique view of TCR signalosome activity solicited from physiologic non-engineered primary human patient samples.

Abstract We have observed Th2 polarization in patients with metastatic malignant melanoma. We hypothesized that changes in the immune system with age may parallel changes we have seen in patients with cancer. We analyzed the peripheral blood of 389 healthy blood donors. We profiled T cell subsets in 389 of the subjects, and antigen-specific tetramers for CMV, influenza and MART-1 in 58 of the subjects. We used a cytokine multiplex to analyze 42 cytokines in 95 of the subjects. We observed significant increases in CD4+ Th2 cells, and decreases in CD3+ T cells, cytotoxic T cells and helper T cells with age. There were fewer CMV-, influenza- and MART-1- specific naïve and CD8+ T cells with age. There were also significant increases in MCP-1 and RANTES with age. We observed differences between males in females in their cytokine profiles in their 20-40’s but not in their 50-60’s. In summary, we have found that aging is associated with an increase in CD4+ Th2 cells and concomitant decreases in total T cell count, cytotoxic T cells and helper T cells. Furthermore, aging is associated with increases in MCP-1 and RANTES. Females had different cytokine profiles than males before menopause, but not after.

Background: Congenital clasped thumb is associated with deficient thumb extensor tendons. Reconstruction includes tendon transfer. Here, we describe a variant of the abductor pollicis longus (APL) tendon, not previously reported, contributing to the flexion deformity. The purpose of this study is to report examples of and offer surgical repair techniques for APL variants identified in patients with clasped thumb. Methods: We reviewed records of 11 consecutive patients undergoing reconstruction for clasped thumb. Surgical anatomy of the APL tendon was evaluated in all patients, followed by release of aberrant APL attachments. Participants were invited to return for an in-person assessment with a certified hand therapist. Data were collected regarding intraoperative findings, surgical techniques for reconstruction, postoperative thumb motion, and patient and caregiver satisfaction. Results: Eleven children (12 thumbs) underwent aberrant APL release and rerouting between 2019 and 2021. Preoperatively, all thumbs were passively correctible to 0° of extension. In all patients, the APL was found to terminate palmar to the metacarpophalangeal (MCP) joint, creating an MCP flexion moment when tensioned. The average age at surgery was 7 years (range: 1-15 years), and the average follow-up was 14.2 months (range: 1-21 months). The mean postoperative thumb radial abduction was 55° (range: 20°-75°). Conclusions: When reconstructing clasped thumbs, surgeons should explore the presence of APL abnormalities. Release and centralization of the APL can improve thumb position and function. This technique may avoid the need for extra-anatomical tendon transfer in patients with clasped thumb.

AbstractBackgroundWhite matter (WM) injury visible on MRI is a common finding in Alzheimer’s disease (AD) and is often attributed to small vessel ischemic changes secondary to increased systemic vascular risk. Increased WM injury has been associated with the progression of Autosomal Dominant AD (ADAD), though ADAD pathogenic variant carriers are relatively young and may not have elevated vascular risk factors. We hypothesized that WM injury in ADAD may reflect worsening of cerebral amyloid angiopathy (CAA) and neurodegeneration. Here we examine this hypothesis using cross‐sectional and longitudinal data from the Dominantly Inherited Alzheimer Network observational study (DIAN).MethodMRI data from ADAD pathogenic variant carriers (n=223) and non‐carriers (n=136) were used in the present study (Table 1). We extracted FreeSurfer‐based WM lesion (WML) volume from T1‐weighted images (hypointensities). Cortical microbleed (CMB) burden was assessed visually on susceptibility weighted/T2*‐weighted gradient echo images by experienced radiologists (blineded to the mutation status) at the Mayo Clinic in Rochester. Linear regression models compared WML volume at baseline in people with and without CMB. Linear mixed effect models assessed the relationships between longitudinal WML and both CMBs and FreeSurfer‐based total gray matter (GM) volume. Models were corrected for age and estimated years to symptom onset (EYO).ResultGreater baseline WML volume was seen in ADAD carriers vs. non‐carriers, particularly close to the age of estimated symptom onset. Baseline WML volume was greater in carriers with CMBs compared to those without (t=2.9, p=0.003, Figure 1). Longitudinal increase in WML amongst ADAD pathogenic variant carriers with CMBs was estimated to be 214 mm3/year greater than that amongst carriers without CMBs (t=4.1, p<0.001, Figure 2). Independent of this CMB effect, decreasing GM volume was strongly associated with increasing longitudinal WML volume (t=‐6.2, p<0.001, Figure 3). Similar analyses in the non‐carrier group yielded no significant findings.ConclusionConsistent with prior reports, WML volume was increased in ADAD pathogenic variant carriers. However, the results here suggest WML in ADAD may not solely be due to small vessel ischemic changes, but rather may be a result of worsening CAA and more rapid neurodegeneration.

AbstractBackgroundIn familial frontotemporal lobar degeneration (f‐FTLD), plasma levels of neurofilament light (NfL) are elevated at least two years prior to symptom onset and predict future clinical status. The Neurofilament Surveillance Project (NSP), an actively recruiting observational study, seeks to obtain higher resolution data of temporal changes in plasma NfL to better understand its clinical utility in f‐FTLD interventional trials. The NSP is a pre‐competitive collaboration sponsored by the Bluefield Project to Cure FTD and funded by biotechnology and pharmaceutical companies and nonprofit organizations.MethodParticipants must be members of families with disease‐causing mutations in C9orf72, MAPT or GRN and enrolled in the ALLFTD natural history study. Up to 335 participants, with roughly equal representation across mutation type, provide plasma quarterly for 3 years (4355 samples total) to enable the observation of peripheral NfL levels longitudinally during disease onset and progression. Plasma is collected by certified mobile research nurses during remote visits, and NfL is measured in batch at least every 6 months. NfL measurements from one plasma aliquot per draw will be correlated with annual assessments obtained in the ALLFTD study, including measures of disease severity (CDRÒ+NACC‐FTLD, CGI, etc.), cognitive function from the UDS Neuropsychology Battery, and volumetric MRI data. Additional plasma aliquots are reserved for future analyses of other biomarkers during the course of the study; another subset of aliquots is reserved to share with interested investigators after the study ends.ResultAs of 1/19/22, the NSP had enrolled 161 participants, including 62 from C9orf72 kindreds, 60 from MAPT kindreds, and 28 from GRN kindreds. One‐hundred forty‐five participants had completed their initial visit, and 109 had completed more than 3, and up to 5, quarterly blood draws. Enrollment and study visits are ongoing despite COVID‐related limitations.ConclusionThe quarterly blood sampling from this study, combined with annual comprehensive clinical assessments through the ALLFTD natural history study, will enable a more detailed understanding of when and how NfL levels change during phenoconversion and/or disease progression in f‐FTLD mutation carriers. These data will be especially informative in the context of the targeted investigational therapeutics for f‐FTLD now entering clinical trials.

AbstractBackgroundAPOE4 gene dose, the number of apolipoprotein E ‐ɛ4 (APOE4) ɛ4 alleles in a person’s genotype, is associated with higher Alzheimer’s disease (AD) risk and younger median age at dementia onset. We previously found relationships between PET, plasma, and measurements of core AD CSF biomarkers in APOE4 gene dose. Here, we characterize longitudinal neurofilament light chain (NfL) changes in the Arizona APOE cohort, including cognitively unimpaired participants, with longitudinal plasma NfL measurements in APOE4 homozygotes (HMs), heterozygotes (HTs) and non‐carriers (NCs).MethodPlasma NfL measurements were performed at the University of Gothenburg using a Single molecule array (Simoa) immunoassay. Annual log‐transformed plasma NfL changes were compared with linear‐tend and univariate ANOVA adjusted for age, sex, and education in 45 HMs, 107 HTs, and 201 NCs who were cognitively unimpaired, 47‐85 years old and did not differ significantly in their age, sex, or educational level. Pearson r correlations with age and annual log‐transformed NfL changes were assessed in 353 participants with at least 2 visits (range 2‐6 visits with an average of ∼3) and an average follow‐up of ∼7 years (range 1‐12 years).ResultAnnual log‐transformed NfL changes were linearly associated with APOE4 gene dose (linear trend HM>HT>NC, P=0.01) and positively associated with age (r = 0.31, P<0.0001). Pairwise comparisons from the univariate ANOVA adjusted for age, sex, and education (P=0.001) showed significantly higher annual NfL changes in HM and HT groups versus NC controls (P=0.01).ConclusionAge and APOE4 gene dose are associated with greater annual plasma NfL changes in CU subjects close to their estimated ages at clinical onset. Studies are needed to better elucidate relationships between different Aβ, tau, inflammatory, and other CSF, or blood‐based biomarkers, and APOE4 gene dose at other ages and in larger subject groups.