CD4+ CAR-T cell exhaustion associated with early relapse of multiple myeloma after BCMA CAR-T cell therapy.

Multiple myeloma is characterized by frequent clinical relapses following conventional therapy. Recently, chimeric antigen receptor T (CAR-T) cells targeting B-cell maturation antigen (BCMA) has been established as a treatment option for patients with relapsed or refractory disease. However, while >70% of patients initially respond to this treatment, clinical relapse and disease progression occur in most cases. Recent studies showed persistent expression of BCMA at the time of relapse, indicating that immune intrinsic mechanisms may contribute to this resistance. While there were no pre-existing T cell features associated with clinical outcomes, we found that patients with a durable response to CAR-T cell treatment had greater persistence of their CAR-T cells compared to patients with transient clinical responses. They also possessed a significantly higher proportion of CD8+ T effector memory cells. In contrast, patients with short-lived responses to treatment have increased frequencies of cytotoxic CD4+ CAR-T cells. These cells expand in vivo early after infusion but express exhaustion markers (HAVCR2 and TIGIT) and remain polyclonal. Finally, we demonstrate that non-classical monocytes are enriched in the myeloma niche and may induce CAR-T cell dysfunction through mechanisms that include TGFß. These findings shed new light on the role of cytotoxic CD4+ T cells in disease progression after CAR-T cell therapy.

A qualitative study of mental health problems among children living in New Delhi slums.

Children living in urban slums in India are exposed to chronic stressors that increase their risk of developing mental disorders, but they remain a neglected group. Effective mental health interventions are needed; however, it is necessary to understand how mental health symptoms and needs are perceived and prioritized locally to tailor interventions for this population. We used an existing rapid ethnographic assessment approach to identify mental health problems from the perspective of children living in Indian slums, including local descriptions, perceived causes, impact, and coping behavior. Local Hindi-speaking interviewers conducted 77 free-list interviews and 33 key informant interviews with children and adults (N = 107) from two slums in New Delhi. Results identified a range of internalizing and externalizing symptoms consistent with depression, anxiety, and conduct problems in children. Findings included both common cross-cultural experiences and symptoms as well as uniquely described symptoms (e.g., "madness or anger," "pain in the heart and mind") not typically included on western standardized measures of psychopathology. Mental health problems appeared to be highly interconnected, with experiences such as harassment and fighting often described as both causes and impacts of mental health symptoms in children. Community perspectives indicated that even in the face of several unmet basic needs, mental health problems were important to the community and counseling interventions were likely to be acceptable. We discuss implications for adapting mental health interventions and assessing their effectiveness to reduce the burden of mental illness among children living in urban slums in India.

Chronic kidney disease and left ventricular diastolic dysfunction (CKD-LVDD) alter cardiac expression of mitochondria-related genes in swine.

Cardiovascular disease and heart failure doubles in patients with chronic kidney disease (CKD), but the underlying mechanisms remain obscure. Mitochondria are central to maintaining cellular respiration and modulating cardiomyocyte function. We took advantage of our novel swine model of CKD and left ventricular diastolic dysfunction (CKD-LVDD) to investigate the expression of mitochondria-related genes and potential mechanisms regulating their expression. CKD-LVDD and normal control pigs (n=6/group, 3 males/3 females) were studied for 14 weeks. Renal and cardiac hemodynamics were quantified by multidetector-CT, echocardiography, and pressure-volume loop studies, respectively. Mitochondrial morphology (electron microscopy) and function (Oroboros) were assessed ex vivo. In randomly selected pigs (n=3/group), cardiac mRNA-, MeDIP-, and miRNA-sequencing (seq) were performed to identify mitochondria-related genes and study their pre- and post -transcriptional regulation. CKD-LVDD exhibited cardiac mitochondrial structural abnormalities and elevated mitochondrial H2O2 emission but preserved mitochondrial function. Cardiac mRNA-seq identified 862 mitochondria-related genes, of which 69 were upregulated and 33 downregulated (fold-change ≥2, false discovery rate≤0.05). Functional analysis showed that upregulated genes were primarily implicated in processes associated with oxidative stress, whereas those downregulated mainly participated in respiration and ATP synthesis. Integrated mRNA/miRNA/MeDIP-seq analysis showed that upregulated genes were modulated predominantly by miRNAs, whereas those downregulated were by miRNA and epigenetic mechanisms. CKD-LVDD alters cardiac expression of mitochondria-related genes, associated with mitochondrial structural damage but preserved respiratory function, possibly reflecting intrinsic compensatory mechanisms. Our findings may guide the development of early interventions at stages of cardiac dysfunction in which mitochondrial injury could be prevented, and the development of LVDD ameliorated.

Adjuvanted SARS-CoV-2 spike protein vaccination elicits long-lived plasma cells in nonhuman primates.

Durable humoral immunity is mediated by long-lived plasma cells (LLPCs) that reside in the bone marrow. It remains unclear whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein vaccination is able to elicit and maintain LLPCs. Here, we describe a sensitive method to identify and isolate antigen-specific LLPCs by tethering antibodies secreted by these cells onto the cell surface. Using this method, we found that two doses of adjuvanted SARS-CoV-2 spike protein vaccination are able to induce spike protein-specific LLPC reservoirs enriched for receptor binding domain specificities in the bone marrow of nonhuman primates that are detectable for several months after vaccination. Immunoglobulin gene sequencing confirmed that several of these LLPCs were clones of memory B cells elicited 2 weeks after boost that had undergone further somatic hypermutation. Many of the antibodies secreted by these LLPCs also exhibited improved neutralization and cross-reactivity compared with earlier time points. These findings establish our method as a means to sensitively and reliably detect rare antigen-specific LLPCs and demonstrate that adjuvanted SARS-CoV-2 spike protein vaccination establishes spike protein-specific LLPC reservoirs.

Tourniquet Use in the Prehospital Setting.

PURPOSE: Tourniquets are a mainstay of life-saving hemorrhage control. The US military has documented the safety and effectiveness of tourniquet use in combat settings. In civilian settings, events such as the Boston Marathon bombing and mass shootings show that tourniquets are necessary and life-saving entities that must be used correctly and whenever indicated. Much less research has been done on tourniquet use in civilian settings compared to military settings. The purpose of this study is to describe the prehospital use of tourniquets in a regional EMS system served by a single trauma center. METHODS: All documented cases of prehospital tourniquet use from 2015 to 2020 were identified via a search of EMS, emergency department, and inpatient records, and reviewed by the lead investigator. The primary outcomes were duration of tourniquet placement, success of hemorrhage control, and complications; secondary outcomes included time of day (by EMS arrival time), transport interval, extremity involved, who placed/removed the tourniquet, and mechanism of injury. RESULTS: Of 182 patients with 185 tourniquets applied, duration of application was available for 52, with a median (IQR) of 43 (56) minutes. Hemorrhage control was achieved in all but two cases (96%). Three cases (5.8%) required more than one tourniquet. Complications included five cases of temporary paresthesia, one case of ecchymosis, two cases of fasciotomy, and two cases of compression nerve injury. The serious complication rate was 7.7% (4/52). Time of day was daytime (08:01-16:00) = 15 (31.9%), evening (16:01-00:00) = 27 (57.4%), and night (00:01- 08:00) = 5 (10.6%). The median transport interval was 22 (IQR 5] minutes. The limbs most often injured were the left and right upper extremities (15 each). EMS clinicians and police officers were most often the tourniquet placers. Common mechanisms of injury included gunshot wounds, motorcycle accidents, and glass injuries. CONCLUSION: Tourniquets used in the prehospital setting have a high rate of hemorrhage control and a low rate of complications.

Durable immunity to SARS-CoV-2 in both lower and upper airways achieved with a gorilla adenovirus (GRAd) S-2P vaccine in non-human primates.

SARS-CoV-2 continues to pose a global threat, and current vaccines, while effective against severe illness, fall short in preventing transmission. To address this challenge, there's a need for vaccines that induce mucosal immunity and can rapidly control the virus. In this study, we demonstrate that a single immunization with a novel gorilla adenovirus-based vaccine (GRAd) carrying the pre-fusion stabilized Spike protein (S-2P) in non-human primates provided protective immunity for over one year against the BA.5 variant of SARS-CoV-2. A prime-boost regimen using GRAd followed by adjuvanted S-2P (GRAd+S-2P) accelerated viral clearance in both the lower and upper airways. GRAd delivered via aerosol (GRAd(AE)+S-2P) modestly improved protection compared to its matched intramuscular regimen, but showed dramatically superior boosting by mRNA and, importantly, total virus clearance in the upper airway by day 4 post infection. GrAd vaccination regimens elicited robust and durable systemic and mucosal antibody responses to multiple SARS-CoV-2 variants, but only GRAd(AE)+S-2P generated long-lasting T cell responses in the lung. This research underscores the flexibility of the GRAd vaccine platform to provide durable immunity against SARS-CoV-2 in both the lower and upper airways.

Online Forums as a Tool for Broader Inclusion of Voices on Health Care Communication Experiences and Serious Illness Care: Mixed Methods Study.

BACKGROUND: Existing health care research, including serious illness research, often underrepresents individuals from historically marginalized communities. Capturing the nuanced perspectives of individuals around their health care communication experiences is difficult. New research strategies are needed that increase engagement of individuals from diverse backgrounds. OBJECTIVE: The aim of this study was to develop a mixed methods approach with qualitative online forums to better understand health communication experiences of individuals, including people from groups historically marginalized such as Black and Latino individuals; older adults; and people with low income, disability, or serious illness. METHODS: We used a multiphase mixed methods, community-informed research approach to design study instruments and engage participants. We engaged a diverse group of collaborators with lived experience of navigating the health care system who provided feedback on instruments, added concepts for testing, and offered guidance on creating a safe experience for participants (phase 1). We conducted a national quantitative survey between April and May 2021 across intrapersonal, interpersonal, and systems-level domains, with particular focus on interpersonal communication between patients and clinicians (phase 2). We conducted two asynchronous, qualitative online forums, a technique used in market research, between June and August 2021, which allowed us to contextualize the learnings and test concepts and messages (phase 3). Using online forums allowed us to probe more deeply into results and hypotheses from the survey to better understand the "whys" and "whats" that surfaced and to test public messages to encourage action around health. RESULTS: We engaged 46 community partners, including patients and clinicians from a Federally Qualified Health Center, to inform study instrument design. In the quantitative survey, 1854 adults responded, including 50.5% women, 25.2% individuals over 65 years old, and 51.9% individuals with low income. Nearly two-thirds identified as non-Hispanic white (65.7%), 10.4% identified as non-Hispanic Black, and 15.5% identified as Hispanic/Latino. An additional 580 individuals participated in online forums, including 60.7% women, 17.4% individuals over 65 years old, and 49.0% individuals with low income. Among the participants, 70.3% identified as non-Hispanic white, 16.0% as non-Hispanic Black, and 9.5% as Hispanic/Latino. We received rich, diverse input from our online forum participants, and they highlighted satisfaction and increased knowledge with engagement in the forums. CONCLUSIONS: We achieved modest overrepresentation of people who were over 65 years old, identified as non-Hispanic Black, and had low income in our online forums. The size of the online forums (N=580) reflected the voices of 93 Black and 55 Hispanic/Latino participants. Individuals who identify as Hispanic/Latino remained underrepresented, likely because the online forums were offered only in English. Overall, our findings demonstrate the feasibility of using the online forum qualitative approach in a mixed methods study to contextualize, clarify, and expound on quantitative findings when designing public health and clinical communications interventions.

What Is Justice? Perspectives of Victims-Survivors of Gender-Based Violence.

This article explores "how do victims-survivors of gender-based violence (GBV) experience and perceive justice?" based on interviews with 251 victims-survivors with experience of different types of GBV and criminal, civil, and family justice systems. Victims-survivors were found to have multiple perceptions of justice, related to different points in their journey following abuse and regarding individual, community, and societal responses. Perceptions relate to accountability; fairness in outcome and process; protection from future harm; recognition; agency; empowerment; affective justice; reparation; and social transformation. Current understandings of justice in legislative and policy approaches reproduce the "justice gap" by failing to take account of how survivors themselves understand and demand justice.

Immune Activation Profiles Elicited by Distinct, Repeated TLR Agonist Infusions in Rhesus Macaques.

TLR agonists are a promising class of immune system stimulants investigated for immunomodulatory applications in cancer immunotherapy and viral diseases. In this study, we sought to characterize the safety and immune activation achieved by different TLR agonists in rhesus macaques (Macaca mulatta), a useful preclinical model of complex immune interactions. Macaques received one of three TLR agonists, followed by plasma cytokine, immune cell subset representation, and blood cell activation measurements. The TLR4 agonist LPS administered i.v. induced very transient immune activation, including TNF-α expression and monocyte activation. The TLR7/8 agonist 2BXy elicited more persistent cytokine expression, including type I IFN, IL-1RA, and the proinflammatory IL-6, along with T cell and monocyte activation. Delivery of 2BXy i.v. and i.m. achieved comparable immune activation, which increased with escalating dose. Finally, i.v. bacillus Calmette-Guérin (BCG) vaccination (which activates multiple TLRs, especially TLR2/4) elicited the most pronounced and persistent innate and adaptive immune response, including strong induction of IFN-γ, IL-6, and IL-1RA. Strikingly, monocyte, T cell, and NK cell expression of the proliferation marker Ki67 increased dramatically following BCG vaccination. This aligned with a large increase in total and BCG-specific cells measured in the lung. Principal component analysis of the combined cytokine expression and cellular activation responses separated animals by treatment group, indicating distinct immune activation profiles induced by each agent. In sum, we report safe, effective doses and routes of administration for three TLR agonists that exhibit discrete immunomodulatory properties in primates and may be leveraged in future immunotherapeutic strategies.

Safety and feasibility of destination care for bariatric surgery: a single institution retrospective study.

INTRODUCTION: Increasing emphasis on value-based healthcare has prompted both employers and healthcare organizations to develop innovative strategies to supply high quality care to patients. One such strategy is through the bundled care payment model (BCPM). Through this model, our institution partnered with employers from across the country to provide quality care for their members. Patients traveling greater than 2 h driving time from the bariatric center were considered "destination" patients. To properly care for our destination patients, our institution created a "destination bariatric program." We sought to investigate comparative outcomes for the first 100 patients who completed the program. We hypothesized that there would be no difference in patient outcomes or complications between destination and local patient groups undergoing sleeve gastrectomy (SG) or Roux-en-Y gastric bypass (RYGB). METHODS AND PROCEDURES: A retrospective cohort analysis of patients undergoing bariatric surgery at a MBSAQIP-accredited bariatric surgery center between May 2019 and October 2021 was conducted. Patients were divided into destination or local patient groups based on participation in the established destination surgery program. Patient demographics, perioperative clinical outcomes, and complications were compared and statistically analyzed using two-sample t-tests, Chi-square tests, Fisher's exact tests, and univariate logistic regressions. RESULTS: This study identified 296 patients, which consisted of destination (n = 110) and local (n = 186) patient cohorts. Patients in the destination group had higher rates of diabetes mellitus (29.1% vs 24.2%, p = 0.029), but otherwise cohorts had similar basic demographics and comorbidities. Outcomes revealed no statistically significant associations between patient cohort (destination versus local) and ED admission (p = 0.305), hospital readmission (p = 0.893), surgical reintervention (p = 0.974), endoscopic-reintervention (p = 0.714), and patient complications in the postoperative period (30 days). CONCLUSION: Participation in destination care programs for bariatric surgery was found to be both safe and feasible. These destination programs represent an opportunity to provide a broader patient population access to complex surgical care.

The impact of interval cholecystectomy timing after percutaneous transhepatic cholecystostomy on post-operative adverse outcomes.

OBJECTIVE: This study aims to explore how timing of interval of cholecystectomy (IC) after percutaneous transhepatic cholecystostomy tube (PTC) placement impacts post-operative outcomes. METHODS: A retrospective database analysis of New York State SPARCs database of IC between 2005 and 2015. The timing for IC ranged between > 1 week and < 2 years. Patients undergoing this procedure were further divided into quartiles using 4-time intervals; 1-5 weeks (Q1), 5-8 weeks (Q2), 8-12 weeks(Q3), and > 12 weeks(Q4). The study's primary outcome was hospital length of stay (LOS). Secondary outcomes included discharge status, 30-day readmission, 30-day ED visit, and 90-day reoperation, surgery type, complication, and bile duct injury. Multivariable regression models were used to compare patients across the four-time intervals after adjusting for confounding factors. RESULTS: A total of 1038 patients with a history of PTC followed by IC between > 1 week and < 2 years were included in the final analysis. The median time to IC was 7.7 weeks. Q2 and Q3 both had a significantly higher median LOS of 3 days versus Q1 and Q4 at median of 5 days (p < 0.0001). Patients from racial and ethnic minorities (e.g., African Americans and Hispanics) were more likely to get their IC after 12 weeks (p < 0.05). Further, Black patients had a significantly higher median LOS than White, non-Hispanic patients (8 days vs 4 days, p < 0.0001) and were more likely to have open procedure. Multivariable regression analysis identified shorter LOS during Q2 (Ratio, 0.76, 95%, 0.67-0.87, p < 0.0001), and Q3 (Ratio 0.75, 95% CI, 065-0.86, p < 0.0001) compared to those who got their IC in Q4. Similar findings exist when comparing Q2 and Q3 to those receiving treatment during Q1. CONCLUSION: A time interval of 5-12 weeks between PTC and IC was associated with a decreased LOS. This study also suggests the persistence of racial disparities among these patients.

The Use of Chemical Control Within Coercive Controlling Intimate Partner Violence and Abuse.

This paper explores the use of chemical control by perpetrators as part of coercive controlling intimate partner violence and abuse, defined as the nonconsenting use of prescribed and nonprescribed medication (including vaccines), and/or other substances to coerce or control, reducing the victim-survivor's capacity for independence, freedom, and health. Based on testimonies of 37 victims-survivors and nine domestic abuse practitioners in the UK we identify varying tactics used to chemically coerce and control, deepening our understanding about the continually changing forms of domestic violence and abuse and enhancing the potential for a more robust response through better informed policy and practice.

RBD-based high affinity ACE2 antagonist limits SARS-CoV-2 replication in upper and lower airways.

SARS-CoV-2 has the capacity to evolve mutations to escape vaccine-and infection-acquired immunity and antiviral drugs. A variant-agnostic therapeutic agent that protects against severe disease without putting selective pressure on the virus would thus be a valuable biomedical tool. Here, we challenged rhesus macaques with SARS-CoV-2 Delta and simultaneously treated them with aerosolized RBD-62, a protein developed through multiple rounds of in vitro evolution of SARS-CoV-2 RBD to acquire 1000-fold enhanced ACE2 binding affinity. RBD-62 treatment gave equivalent protection in upper and lower airways, a phenomenon not previously observed with clinically approved vaccines. Importantly, RBD-62 did not block the development of memory responses to Delta and did not elicit anti-drug immunity. These data provide proof-of-concept that RBD-62 can prevent severe disease from a highly virulent variant.

Trispecific antibody targeting HIV-1 and T cells activates and eliminates latently-infected cells in HIV/SHIV infections.

Agents that can simultaneously activate latent HIV, increase immune activation and enhance the killing of latently-infected cells represent promising approaches for HIV cure. Here, we develop and evaluate a trispecific antibody (Ab), N6/αCD3-αCD28, that targets three independent proteins: (1) the HIV envelope via the broadly reactive CD4-binding site Ab, N6; (2) the T cell antigen CD3; and (3) the co-stimulatory molecule CD28. We find that the trispecific significantly increases antigen-specific T-cell activation and cytokine release in both CD4+ and CD8+ T cells. Co-culturing CD4+ with autologous CD8+ T cells from ART-suppressed HIV+ donors with N6/αCD3-αCD28, results in activation of latently-infected cells and their elimination by activated CD8+ T cells. This trispecific antibody mediates CD4+ and CD8+ T-cell activation in non-human primates and is well tolerated in vivo. This HIV-directed antibody therefore merits further development as a potential intervention for the eradication of latent HIV infection.

Computational Methods for Single-Cell Proteomics.

Advances in single-cell proteomics technologies have resulted in high-dimensional datasets comprising millions of cells that are capable of answering key questions about biology and disease. The advent of these technologies has prompted the development of computational tools to process and visualize the complex data. In this review, we outline the steps of single-cell and spatial proteomics analysis pipelines. In addition to describing available methods, we highlight benchmarking studies that have identified advantages and pitfalls of the currently available computational toolkits. As these technologies continue to advance, robust analysis tools should be developed in tandem to take full advantage of the potential biological insights provided by these data.

Dynamic CD8+ T cell responses to cancer immunotherapy in human regional lymph nodes are disrupted in metastatic lymph nodes.

CD8+ T cell responses are critical for anti-tumor immunity. While extensively profiled in the tumor microenvironment, recent studies in mice identified responses in lymph nodes (LNs) as essential; however, the role of LNs in human cancer patients remains unknown. We examined CD8+ T cells in human head and neck squamous cell carcinomas, regional LNs, and blood using mass cytometry, single-cell genomics, and multiplexed ion beam imaging. We identified progenitor exhausted CD8+ T cells (Tpex) that were abundant in uninvolved LN and clonally related to terminally exhausted cells in the tumor. After anti-PD-L1 immunotherapy, Tpex in uninvolved LNs reduced in frequency but localized near dendritic cells and proliferating intermediate-exhausted CD8+ T cells (Tex-int), consistent with activation and differentiation. LN responses coincided with increased circulating Tex-int. In metastatic LNs, these response hallmarks were impaired, with immunosuppressive cellular niches. Our results identify important roles for LNs in anti-tumor immune responses in humans.

[S073]-The impact of interval appendectomy timing on follow-up adverse outcomes.

OBJECTIVE: This study aims to compare the timing of interval appendectomy (IA) and its impact on post-operative outcomes. METHODS: A retrospective analysis was performed for adult patients diagnosed with appendicitis between 2006 and 2017. IA was defined as a follow-up appendectomy > 1 week and < 2 years after the initial presentation. Time intervals were divided into 4 groups based on patient quartiles: 1-6 weeks, 7-9 weeks, 10-15 weeks, and > 15 weeks. The primary outcome measure was length of stay (LOS). Secondary outcomes included 30-day readmission and IA post-operative complications. Tertiary outcomes included 30-day mortality and colonoscopy suggesting neoplasm or Inflammatory Bowel Disease. RESULTS: A total of 5069 patients' records whose interval appendectomy fell > 1 week and < 2 years after initial presentation were analyzed. Among them, 1006 (19.85%) underwent an initial percutaneous abscess drainage at diagnosis. The median timing for IA was 9.2 weeks. Patients with IA at 1-6 weeks were more likely to have longer LOS when compared to 7-9 weeks (ratio 1.33, 95% CI 1.2-1.48) and 10-15 weeks (ratio 1.38, 95% CI 1.25-1.52). IA between 7 and 9 weeks (ratio 0.81, 95% CI 0.73-0.89) and 10-15 weeks (ratio 0.78, 95% CI 0.71-0.86) was associated with significantly shorter LOS compared to those receiving the operation after 15 weeks. Further, patients requiring abscess drainage (ratio 1.2, 95% CI 1.13-1.34) or those with comorbidities (ratio 1.51, 95% CI 1.39-1.63) were more likely to have longer LOS at IA. Socioeconomic and demographic differences including Black, Hispanic, and those with Medicare and Medicaid insurance had a greater LOS after their IA. CONCLUSION: LOS remains lowest among patients undergoing IA between 7-9 weeks and 10-15 weeks after initial appendicitis presentation. Patients with lower socioeconomic status or from racial minorities had a longer LOS after IA.

Prolonged Hyperglycemia Causes Visual and Cognitive Deficits in Danio rerio.

The present study induced prolonged hyperglycemia (a hallmark symptom of Type 2 diabetes [T2DM]) in Danio rerio (zebrafish) for eight or twelve weeks. The goal of this research was to study cognitive decline as well as vision loss in hyperglycemic zebrafish. Fish were submerged in glucose for eight or twelve weeks, after which they were assessed with both a cognitive assay (three-chamber choice) and a visual assay (optomotor response (OMR)). Zebrafish were also studied during recovery from hyperglycemia. Here, fish were removed from the hyperglycemic environment for 4 weeks after either 4 or 8 weeks in glucose, and cognition and vision was again assessed. The 8- and 12-week cognitive results revealed that water-treated fish showed evidence of learning while glucose- and mannitol-treated fish did not within the three-day testing period. OMR results identified an osmotic effect with glucose-treated fish having significantly fewer positive rotations than water-treated fish but comparable rotations to mannitol-treated fish. The 8- and 12-week recovery results showed that 4 weeks was not enough time to fully recovery from the hyperglycemic insult sustained.

Novel AAV-mediated genome editing therapy improves health and survival in a mouse model of methylmalonic acidemia.

Methylmalonic acidemia (MMA) is an inborn error of metabolism mostly caused by mutations in the mitochondrial methylmalonyl-CoA mutase gene (MMUT). MMA patients suffer from frequent episodes of metabolic decompensation, which can be life threatening. To mimic both the dietary restrictions and metabolic decompensation seen in MMA patients, we developed a novel protein-controlled diet regimen in a Mmut deficient mouse model of MMA and demonstrated the therapeutic benefit of mLB-001, a nuclease-free, promoterless recombinant AAV GeneRideTM vector designed to insert the mouse Mmut into the endogenous albumin locus via homologous recombination. A single intravenous administration of mLB-001 to neonatal or adult MMA mice prevented body weight loss and mortality when challenged with a high protein diet. The edited hepatocytes expressed functional MMUT protein and expanded over time in the Mmut deficient mice, suggesting a selective growth advantage over the diseased cells. In mice with a humanized liver, treatment with a human homolog of mLB-001 resulted in site-specific genome editing and transgene expression in the transplanted human hepatocytes. Taken together, these findings support the development of hLB-001 that is currently in clinical trials in pediatric patients with severe forms of MMA.

Circulating monocytes associated with anti-PD-1 resistance in human biliary cancer induce T cell paralysis.

Suppressive myeloid cells can contribute to immunotherapy resistance, but their role in response to checkpoint inhibition (CPI) in anti-PD-1 refractory cancers, such as biliary tract cancer (BTC), remains elusive. We use multiplexed single-cell transcriptomic and epitope sequencing to profile greater than 200,000 peripheral blood mononuclear cells from advanced BTC patients (n = 9) and matched healthy donors (n = 8). Following anti-PD-1 treatment, CD14+ monocytes expressing high levels of immunosuppressive cytokines and chemotactic molecules (CD14CTX) increase in the circulation of patients with BTC tumors that are CPI resistant. CD14CTX can directly suppress CD4+ T cells and induce SOCS3 expression in CD4+ T cells, rendering them functionally unresponsive. The CD14CTX gene signature associates with worse survival in patients with BTC as well as in other anti-PD-1 refractory cancers. These results demonstrate that monocytes arising after anti-PD-1 treatment can induce T cell paralysis as a distinct mode of tumor-mediated immunosuppression leading to CPI resistance.