Citations

Abstract Introduction The H-2Db class I restricted CD8 T cell response is critical for clearance of neurotropic Theiler’s virus encephalomyelitis virus (TMEV) infection. However, the key central nervous system cell types required to present H-2Db for CD8 T cell trafficking and expansion in the brain are not known. Mural cells are a critical cellular component of the neurovascular unit for maintaining vascular integrity which also express MHC class I molecules. However, the importance of mural cell antigen-restricted engagement by CD8 T cells has not been investigated. Methods To determine the function of mural cell MHC Class I antigen presentation, we crossed the Pdgfrβ-creERT2 mouse to our novel H-2Db LoxP mouse developed by our laboratory. This transgenic mouse approach enabled us to target mural cells during neuroinflammation. Results We observed robust knockdown of H-2Db on mural cells during acute TMEV infection. Conditional H-2Db knockdown in mural cells resulted in increased brain infiltration of CD8 T cells specific to the immunodominant Db:VP2 121-130 epitope of TMEV, demonstrating an important role of mural cells for immune cell recruitment. Conclusion To our knowledge, this is the first study demonstrating MHC class I antigen presentation by mural cells impacts CD8 T cell recruitment during neuroinflammation. Funding Source R01-NS103212, T32-AI170478 Topic Categories Neuroimmunology (NEUR)

Abstract Introduction T cell activation is a dynamic process governed by the strength and duration of antigen recognition via the T cell receptor (TCR). The timing of in vivo TCR signaling— when and how strongly memory CD8+ T cells become reactivated in the inflamed brain following antigen exposure— is a fundamental mechanism that remains unclear. The Nr4a3-Tocky reporter (Tocky) mouse employs a fluorescent timer that shifts from blue to red after TCR-driven Nr4a3 activation, which allows researchers to track activation kinetics through two parameters: the Timer Angle (recency of T cell activation), and the Timer Intensity (strength of signaling). Methods To understand CD8+ T cell activation during this neuroinflammation, we generated memory CD8+ T cells by intracranial Theiler’s murine encephalomyelitis virus infection of these Tocky mice. After viral clearance, we reactivated memory CD8+ T cells by intravenously injecting their cognate antigen. Tissues were collected 0-72 hours post-injection and profiled by flow cytometry. An R pipeline was constructed to transform event-level flow data to quantify the TCR activation. Data were analyzed with a Metropolis—Hastings MCMC algorithm to model activation dynamics and estimate probabilistic transitions between biologically defined states (newly activated, persistent, arrested states). Results These empirical transition probabilities enabled Bayesian comparison of antigen-specific versus nonspecific CD8+ T cells. It was revealed that antigen-specific CD8+ T cells rapidly transition to a high-intensity activation state within 24 hours and maintain persistent signaling compared to nonspecific populations. Conclusion This establishes a biologically grounded, in vivo framework for modeling and comparing TCR signaling during neuroinflammation. Funding Source R01 - NS103212 Topic Categories Neuroimmunology (NEUR)

Abstract Introduction Glioblastoma (GBM) is the most aggressive primary brain tumor in adults with an incident rate of 12,000 cases in the United States every year. Current treatment options for these patients include surgical resection, radiation, and chemotherapy. Despite these conventional treatments, patients have a dismal prognosis mostly due to the inability to deliver therapeutic agents across the blood-brain barrier. In recent years, the use of oncolytic picornavirus has been employed as a novel virotherapy for GBM. We hypothesized that we could enhance antibody delivery to the brain through harnessing the immune response associated with this oncolytic virus. Prior work from our lab has shown that CD8 T cell engagement with MHC class I on brain endothelium induces CNS vascular permeability which is dependent on perforin. We therefore asked if picornavirus antigen specific CD8 T cells could enhance antibody delivery to the brain. Methods To test this, we evaluated the potential of activating MHC class I restricted CD8 T cells in vivo through intravenous administration of their cognate picornavirus antigens. Results We determined that this approach enhanced delivery of antibody to brain parenchyma. Importantly, antibody delivery to the brain was still enhanced when CD8 T cells lacked perforin expression which significantly reduced the toxicity of this combination approach. Conclusion These findings demonstrate that theCD8 T cell response towards virotherapy can be harnessed to promote delivery of therapeutic antibody to the brain. Funding Source NCI — R01 CA 94953 NINDS — R01 NS122174 NINDS —R01 NS103212 Mayo Clinic Graduate School of Biomedical sciences Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Abstract Introduction CD8 T cells are key immune cells in viral clearance, targeting infected cells following recognition of MHC class I restricted antigen presentation. Previous work has determined that microglia and perivascular macrophages are essential for normal CD8 T cell responses against neurotropic viral infection, indicating that cells at the neurovascular unit can act as antigen-presenting cells. However, the specific importance of microglia MHC class I restricted antigen presentation to brain-infiltrating CD8 T cells has been difficult to define. Methods To address this question, our laboratory generated novel single MHC Class I conditional knockout mice in which H-2Kb or H-2Db can be deactivated specifically in Tmem119+ microglia with tamoxifen administration. Recombinant Theiler’s murine encephalomyelitis virus (TMEV) encoding the model OVA antigen enabled analysis of virus antigen-specific CD8 T cells restricted to H-2Kb class I molecules while Daniel’s strain TMEV H-2Db class I molecules enabled analysis of Db:VP2 specific CD8 T cells. Results Our results revealed profound differences in the response of CD8 T cells upon deletion of MHC class I molecules on microglia. Conditional knockout of H-2Kb in Tmem119+ microglia reduced Kb:OVA epitope specific CD8 T cells in the brain compared to Cre negative littermate controls, and BrdU staining revealed differences in T cell proliferation. Meanwhile, mice with deletion of Db in Tmem119+ microglia had reduced levels of perforin in Db:VP2 CD8 T cells. Furthermore, in a model of CNS vascular disease, the deletion of H-2Db of Tmem119+ microglia reduced CD8 T cell numbers in the brain, as well as the blood-brain barrier (BBB) breakdown observed by a FITC albumin assay. Conclusion These findings demonstrate discrete roles for specific MHC Class I molecules on microglia during CNS viral infection. This data will further our understanding of brain infiltrating CD8 T cell responses in neurologic diseases as well as the regulation of BBB by microglia. Funding Source R01 NS 103212 R01 NS122174 Topic Categories Neuroimmunology (NEUR)

Background: Itraconazole is the preferred antifungal agent for mild to moderate histoplasmosis and step-down therapy following amphotericin B treatment. Although hydroxy-itraconazole, a major metabolite of itraconazole, displays antifungal activity comparable with that of the parent compound, its role in therapeutic drug monitoring targets for histoplasmosis remains uncertain. Methods: The objective was to compare clinical outcomes and adverse events using itraconazole parent-compound concentrations alone versus combined itraconazole and hydroxy-itraconazole concentrations in patients treated for histoplasmosis. This retrospective cohort study included adults with probable or proven histoplasmosis who received itraconazole and had at least 1 documented serum concentration. The primary outcome was clinical response across 3 groups: (1) parent (itraconazole parent-compound concentration >1.0 mcg/mL), (2) combined (parent-compound concentration <1.0 mcg/mL with combined itraconazole plus hydroxy-itraconazole >1.0 mcg/mL), and (3) subtherapeutic (failure to reach either threshold). Treatment response and adverse events were analyzed using the Fisher exact test. Results: Among 108 patients (parent, n = 40; combined, n = 40; subtherapeutic, n = 28), response rates differed across groups (parent (95%); combined (90%); subtherapeutic (67.9%); P = 0.006). There was no difference between the parent and combined groups. Adverse events did not differ, although drug discontinuation occurred more frequently in the subtherapeutic group (parent (5%); combined (20%); subtherapeutic (25%); P = 0.04). Conclusions: Patients who failed to achieve either an itraconazole concentration >1 mcg/mL or a combined itraconazole plus hydroxy-itraconazole concentration >1 mcg/mL had lower response rates. Similar outcomes among patients achieving the itraconazole target alone and those achieving the combined target suggest that hydroxy-itraconazole concentrations may contribute to therapeutic drug monitoring targets in histoplasmosis.

Abstract Introduction The anatomical locations of generating adaptive immune responses to neurotropic viruses are still being investigated. Skull bone marrow (BM) has recently been identified as a pivotal immune site that can sense and respond to a local insult in the brain. Yet, there is a lack of understanding of the role of skull BM during viral encephalitis. We hypothesized that the skull BM is an important niche for antiviral CD8 T cells priming, expansion, and maintenance during Theiler’s murine encephalomyelitis virus infection. Methods Using peptide:MHC tetramer analysis, we determined that antiviral CD8 T cells are primed in the skull BM in early infection, at time points comparable to cervical lymph nodes. Employing MHC class I conditional knockout mice generated by our research group, we inactivated H-2Kb and H-2Db class I molecules on discrete antigen-presenting cell types. Results We determined using these transgenic mice that viral antigen presentation on dendritic cells (DCs) and endothelial cells is essential for the generation of virus-specific T cells in skull BM during acute viral encephalitis. However, antigen presentation by DCs is not required for long-lived antiviral CD8 T cells that reside in skull BM. Moreover, the use of FTY720 to block lymphocyte migration diminished skull antiviral CD8 T cell numbers, suggesting that peripheral trafficking is required for generating skull antiviral CD8 T cells. Conclusion We present compelling evidence for the importance of skull BM as a responsive site that is highly suitable for rapid generation of antiviral CD8 T cells during acute viral encephalitis. Skull BM also serves as a reservoir for memory T cells, long after the resolution of a neurotropic virus infection. This work sheds light on the skull as a potential target for further modulation of the immune response to viral infections in the brain. Funding Source NIH grants R01 NS5103212 and RF1 NS122174 Topic Categories Viral Immunology (VIR)

ABSTRACT The recently published BLAST clinical risk model for chronic myelomonocytic leukemia (CMML) and its molecular version (BLAST‐Mol) differentiate low, intermediate, and high‐risk groups. The objective of the current retrospective study was to examine the survival impact of allogeneic stem cell transplant (ASCT) in a consecutive series of Mayo Clinic patients ( n = 775; 68% males, median age 71 years) with CMML, overall and adjusted for baseline BLAST/BLAST‐Mol risk categories. At a median follow‐up of 77 months, 151 (20%) patients underwent ASCT. Overall survival (OS) was longer in the ASCT compared to the non‐ASCT cohorts (median 77 vs. 28 months; p < 0.01). The significant difference in OS between the ASCT and non‐ASCT cohorts was retained after adjusting for BLAST clinical risk groups: high‐risk (median 50 vs. 14 months; p < 0.01), intermediate‐risk (81 vs. 28 months; p < 0.01), and low‐risk (111 vs. 65 months; p = 0.04), respectively. The same was mostly the case when OS comparison was adjusted for BLAST‐Mol with respective p values of < 0.01, < 0.01, and 0.08, respectively. Time‐dependent cox models confirmed the favorable survival impact of ASCT in the BLAST/BLAST‐Mol high‐ and intermediate‐risk groups. The current study highlights the benefit of ASCT for BLAST/BLAST‐Mol high‐ and intermediate‐risk groups in CMML and its potential to overcome the adverse impact of some high risk cytogenetic or molecular abnormalities. These observations support the early use of ASCT in CMML, ideally before BT and regardless of genetic characteristics.

Background The management of osteochondral lesions of the talus (OLT) is challenging, with 2 main options being conservative and surgical management. Orthobiologic therapies come to bridge the gap between these 2 options; however, inconsistencies in indication, composition, and heterogeneous evidence limit clinical decision-making. To address these challenges, 3 International Congress Meetings on Cartilage Repair of the Ankle were convened in Pittsburgh, USA (2017), Dublin, Ireland (2019), and New York, USA (2025) to establish agreement on the role of conservative management and injectable orthobiologic strategies in the treatment of OLT. Methods Seventy-five multidisciplinary experts from over 25 countries and 1 territory participated in a structured Delphi-based consensus process. Blinded surveys, structured questionnaires, and in-person discussions were used to develop and refine statements, supported by literature review and graded evidence (A1–E). Consensus strength was defined as consensus (51%-74%), strong consensus (75%-99%), or unanimous (100%) Results In total, 29 consensus statements were achieved across the 3 meetings. Strong agreement was reached regarding indications and expected outcomes of conservative management, including the role of immobilization and appropriate timing for surgical intervention. Orthobiologic injections, including platelet-rich plasma, concentrated bone marrow aspirate, and hyaluronic acid, were considered appropriate in select patients with persistent symptoms, although no superiority was demonstrated among formulations, preparation methods, or injection strategies. When used as adjuncts to surgical procedures, orthobiologics may improve radiological outcomes, with limited evidence for improvement in clinical outcomes. Technical considerations, including preferred harvest sites and a lack of standardized preparation protocols, were identified, along with key principles for patient counseling Conclusion These international consensus statements establish a standardized framework for the conservative management and use of injectable orthobiologic therapies in OLT. By clarifying indications, technical considerations, and current limitations, this work aims to improve consistency in clinical practice, guide treatment decision-making, and inform future research.

Abstract Early-phase clinical trials of Bruton’s tyrosine kinase (BTK) degraders have demonstrated efficacy in patients with BTK inhibitor-resistant chronic lymphocytic leukemia (CLL). How clinical resistance to BTK degraders arises is unknown. Here we sequenced serial CLL samples from patients enrolled in the phase I trials of zelebrudomide and bexobrutideg and observed recurrent expansion of preexisting BTK A428D mutations at relapse. Unlike previously studied BTK inhibitor resistance mutations, BTK A428D conferred pan-resistance to BTK inhibitors and degraders. In the absence of BTK-directed therapies, however, cells bearing BTK A428D exhibited a competitive disadvantage. A crystal structure of BTK A428D revealed that the mutant aspartate clashes with the adenine ring of ATP and the adenine-mimetic moiety of BTK inhibitors and degraders. Combining BTK degraders with venetoclax mitigated the expansion of BTK A428D. These results provide the molecular basis for clinical resistance to BTK degraders and will inform the development of next-generation BTK degrader therapies.