Citations

Abstract Description The importance of microglia class I restricted antigen presentation to brain-infiltrating CD8 T cells has been difficult to define. 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 antigen OVA enabled analysis of virus antigen-specific CD8 T cells restricted to Kb class I molecules while Daniel’s strain TMEV Db class I molecules enabled analysis of Db:VP2 specific CD8 T cells. Our results revealed profound differences between MHC class I molecules. Conditional knockout of Kb in Tmem119+ microglia reduced Kb:OVA epitope specific CD8 T cells in the brain compared to Cre negative littermate controls. Employing methodology using FTY720 and BrdU labeling, we determined that deletion of Kb in Tmem119+ microglia reduced proliferation and numbers of Kb:OVA specific CD8 T cells in the brain compared to Cre negative littermate controls. Meanwhile, mice with deletion of Db in Tmem119+ microglia had reduced levels of perforin in Db:VP2 CD8 T cells. These findings demonstrate there are discrete roles for specific MHC Class I molecules on microglia during CNS viral infection. These results plus transgenic mouse systems will help further our understanding of brain infiltrating CD8 T cell responses in neurologic disease. Funding Sources Supported by R01NS122174 and R01NS103212 Topic Categories Neuroimmunology (NEUR)

Abstract Description Tauopathies are a subset of neurological diseases associated with pathologic tau protein accumulation and the formation of neurofibrillary tangles in the brain. There is growing evidence of immune cell infiltration including T cells and an increase of inflammatory microglia in human brains from patients that have Tauopathies. However, the effect of T cells and how they interact with CNS-resident immune cells in these pathologies is not well understood. To answer this question, we developed a neurotropic AAV1 encoding human mutant P301L-Tau. AAV-Tau transduction of neurons in neonatal mice recapitulates features of Tauopathies found in human patients. As microglia are the predominant resident antigen presenting cells within the CNS, we hypothesized that CD8 T cells may be dependent on MHC class I presentation by microglia in mice with Tauopathy to achieve full infiltration and expansion in the brain. To address this hypothesis, we introduced AAV-Tau into our novel transgenic mice for which MHC class I antigen presentation by H-2Kb on microglia can be conditional ablated. We observed lower frequencies of CD8 T cell populations and less CD8 T cell CD69+CD103+ expression in mice with Tauopathy when MHC class I was ablated in microglia. We conclude that microglia class I restricted antigen presentation contributes to the expansion of resident memory CD8 T cells in experimental Tauopathy and therefore are an important driver of the immune response observed in this dementia model. Funding Sources NIH/NINDS: R01NS122174 Mayo Graduate School of Biomedical Sciences Topic Categories Neuroimmunology (NEUR)

Abstract Description Blood-brain barrier (BBB) disruption is a putative mechanism of neurodegeneration, neuropsychiatric disorders, and long covid. Our laboratory was the first to demonstrate antigen-specific CD8 T cells induce BBB disruption. We now introduce a novel neurologic disease model in which CD8 T resident memory (TRM) cells induce BBB disruption via administration of cognate antigen, long after neurotropic virus is cleared. C57BL/6 mice intracranially infected with Theiler’s murine encephalomyelitis virus (TMEV) clear virus within 21 days. At 2 months post infection, mice received intravenous injection of viral peptide VP2(121-130) for which the largest population of CD8 TRM have T cell receptor specificity for, resulting in BBB permeability detectable by gadolinium enhanced T1 weighted MRI. A second round of BBB disruption was then initiated with VP2 peptide at 4 months post infection. One month after this second induction of BBB disruption, mice underwent behavioral assessments and whole brain flow cytometric analysis. In these mice, we found a significant inverse correlation of BBB breakdown with a shift from active to passive anxiety-like behavior to contextual fear memory compared to mock peptide controls. This shift in anxiety correlated with virus antigen specific CD8 TRM, myeloid, and NK cells. These results demonstrate memory antiviral CD8 T cells can induce BBB disruption, long after virus is cleared, a process that contributes to a shift in anxiety-like behaviors Funding Sources RO1 NS103212, R01 NS122174 Topic Categories Neuroimmunology (NEUR)

Abstract Description Brain Barrier (BBB) disruption plays a role in various neurodegenerative diseases such as ALS, stroke, Huntington’s disease and Cerebral Malaria. In our laboratory, we aim to interrogate the immunological underpinnings associated with neuroinflammation and barrier leakage. In clinical studies, aging is associated with a decline in T cell production and a gradual shift to resident memory T cells in the brain characterized by reduced T cell repertoire diversity and weak activation. To model these clinical observations, we sought to define the effect of MHC class I restricted antigen presentation on endothelial cells of brain vasculature on CD8 T cell infiltration of the CNS. We conditionally ablated H-2Kb and H-2Db molecules on endothelial cells using Cdh5-Cre mice crossed with our novel Kb LoxP and Db LoxP mice generated by our research program. In these animals we studied CD8 T cell accumulation in the brain using Theiler’s Murine Endothelial Virus (TMEV) infection of young mice (<12 months) compared to old mice (>12 months). Our results indicate that the loss of endothelial cell Kb induces a defect in establishment of antigen experienced CD8 T cell populations in acute infection. These finding will allow us to further investigate the role of endothelial cell MHC Class I molecules in the immune response to chronic TMEV infection, as well as other neurologic disease models. Funding Sources NIH Grants R01 NS103212 and R01 NS122174 Topic Categories Neuroimmunology (NEUR)

Abstract Description Signal regulatory protein-α (SIRPα) is a key member of the “do-not-eat-me” signaling pathway and is expressed on intratumoral monocytes/macrophages in lymphoma. In this study, we found that SIRPα is also expressed by a subset of malignant B cells in lymphoma. SIRPα+ B cells have a memory phenotype with germinal center (GC) dark-zone features, and RNA velocity analysis showed that SIRPα+ B cells exhibit higher spliced transcripts confirming a more mature phenotype. SIRPα expression is substantially upregulated by treating lymphoma B cells with LPS or CpG and SIRPα+ B cells showed increased proliferation when compared to SIRPα- B cells, which was associated with reduced cellular autophagy. SIRPα+ B cells exhibited increased expression of BAFF signaling pathway molecules as the likely mechanism of increased proliferation and survival. Furthermore, in co-culture, SIRPα+ lymphoma B cells suppressed T-cell function and blocking SIRPa/CD47 signaling reversed T cell inhibition. Clinically, we found that a greater number of SIRPα+ B cells present in the diagnostic tumor biopsies of FL patients was associated with a significantly poorer overall survival. We conclude that SIRPα+ lymphoma B cells are a proliferative component of indolent lymphomas, are associated with T-cell suppression, and increased prevalence of these cells is associated with a poor prognosis. Targeting SIRPα+ B cells in lymphoma may therefore be a promising therapeutic strategy. Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Description The efficacy of immunotherapy for splenic marginal zone lymphoma (SMZL) remains limited due to the strong immune suppression present in the tumor microenvironment (TME). Tregs play a key role in maintaining TME homeostasis by suppressing effector T cells and malignant B cells. Previously, we identified two Treg subsets CD26+ and CD161+ in SMZL. CD161+ Tregs were more effective at suppressing both CD8+T cells and malignant B cells, whereas CD26+Tregs primarily inhibited CD8+T cells. Patients with higher levels of CD26+ Tregs and fewer CD161+ Tregs tend to have worse outcomes. In this study, we found that cytokine-inducible SH2-containing protein (CISH), a known negative regulator of immune activation, is highly expressed in CD26+Tregs compared to CD161+ Tregs. Co-culturing B cells enhanced the expression of CISH and CD26 in CD4+CD25- Treg cells, knockdown of CISH in Tregs reduced CD26 expression and their immune suppression activity of CD8+T cells, while CRISPR knockout of CISH in Karpas 299 cells led to higher expression CD70, a molecule associated with less immunesuppression. Our findings suggest that increased CISH expression in Tregs could result in increased immune suppression and unfavorable outcome in SMZL, suggesting CISH is a potential therapeutic tagrget in SMZL by dempening immunosuppressive property of the Treg cells hence boosting the antitumor immunity. Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Description CD8 T cell induced blood-brain barrier (BBB) disruption is an established mechanism of neuropathology. The pericyte is a MHC class I expressing neurovascular unit cell type which is critical for maintaining BBB integrity. However, the importance of pericyte antigen-restricted engagement by CD8 T cells is not fully understood. To determine the function of pericyte 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 pericytes during neuroinflammation. We observed robust knockdown of H-2Db on Pdgfrβ+ pericytes during TMEV infection in these animals. The effect of conditional H-2Db knockdown on this cell type resulted in increased CD4 T cell, CD8 T cell, and B cells infiltration of the brain during TMEV infection, demonstrating an important role of Pdgfrβ+ pericytes for immune cell recruitment into the CNS. To our knowledge, this is the first study demonstrating an important role for MHC class I antigen presentation by pericytes in a murine model of neuroinflammation. Funding Sources Supported by R01NS122174 and R01NS103212 Topic Categories Neuroimmunology (NEUR)

Abstract Description The dura mater of the meninges recruits immune cells for CNS surveillance and response to infections, serving as a critical interface between the brain and the peripheral immune system. Theiler’s murine encephalomyelitis virus (TMEV) infection is a model neurotropic virus for which MHC class I molecules are critical for sterilizing immunity. Using spectral flow cytometry, we profiled the immune response in the dura mater in healthy and TMEV-infected wildtype mice. Acute TMEV infection resulted in the infiltration of diverse immune cell types into the dura mater, including increased cDC1, pDCs, monocytes, NK cells, CD4 T cells, and CD8 T cells. We next examined the importance of MHC class I molecules on diverse immune cell populations in the dura mater during acute TMEV infection. MHC class I deficiency resulted in increased numbers of CD4 T cells with high expression of PD1 in MHC class I-/- (H-2Kb-/-, H-2Db-/-) mice compared to WT mice 7 days post TMEV infection. This demonstrated the importance of MHC class I and CD8 T cells in modulating dura immune cell populations and implies a compensatory shift toward a CD4 T cell response during acute infection. Subsequent studies employing in vivo cell-specific ablation of MHC class I molecules in discrete cell types will further define the impact of antigen presentation on immune regulation and inflammatory outcomes within the dura mater. Topic Categories Neuroimmunology (NEUR)

Importance Total pancreatectomy (TP) is indicated for advanced pancreatic cancer or multifocal tumors. Furthermore, TP may be performed to avoid the risk of pancreatic fistula in selected patients to improve the perioperative risk profile. Objective To define reference values for TP based on a low-risk cohort treated at expert centers. Design, Setting, and Participants This multicenter study analyzed outcomes from patients undergoing primary TP for malignant or benign lesions from 25 international expert centers from January 2017 to November 2023. Low-risk patients undergoing TP (LR-TP) were without vascular resections or significant comorbidities. Exposures TP. Main Outcomes and Measures Twenty reference values were derived from the 75th or the 25th percentile of the median values of all centers. Outcomes of LR-TP were compared with a cohort of TP with vascular resection, TP due to high-risk pancreatic anastomosis, and the benchmark values for low-risk pancreatoduodenectomy. Results Of 994 patients, 333 (33.5%; median [IQR] age, 66 [58-72] years; 171 male [51.4%]) qualified as the LR-TP cohort. Reference values included blood loss (≤1000 mL), major complications (≤37%), 3-month postoperative mortality (<6%), and retrieved lymph nodes (≥29). Compared with TP with vascular resections, reference cutoffs were not met for major complications (51% vs LR-TP ≤37%) and 90-day mortality (11% vs LR-TP ≤6%). For TP due to high-risk anastomosis, failure to rescue rate (38% vs ≤6%) and 90-day mortality (11% vs LR-TP ≤6%) were not met. Compared with pancreatoduodenectomy, reference values for postoperative mortality were 3 times higher for LR-TP (≤2% vs ≤6%) and less for resected lymph nodes (≥16 vs ≥29). Conclusions and Relevance This case-control study provided global reference values for TP, indicating significantly higher postoperative morbidity and mortality compared with pancreatoduodenectomy. Perioperative morbidity of TP was especially increased in patients with vascular resections. These reference values can serve for quality control of pancreatic surgery.

ABSTRACT Background We recently published the first real‐world multicenter and multi‐institutional study of cisplatin, gemcitabine, and durvalumab in patients with advanced biliary tract cancer (BTC). Here we present an expanded patient cohort with an increased sample size and longer median follow‐up. Methods The study population included patients with advanced BTC, who received cisplatin/gemcitabine plus durvalumab at 55 centers across 12 countries in Europe, the United States, and Asia. The primary endpoints of the study were progression‐free survival (PFS) and overall survival (OS). Secondary endpoints were overall response rate (ORR) and safety. Results Overall, 1358 patients were enrolled. Median PFS was 7.6 months (95% CI: 7.2–8.1), and median OS was 15.6 months (95% CI: 14.8–16.5). ORR was 35.6%, and DCR was 82.7%. Any grade AEs occurred in 1213 patients (89.3%). Grade 3–4 AEs occurred in 597 patients (43.2%). The rate of immune‐related AEs (irAE) was 20.3%. Grade 3–4 irAE occurred in 3.0% of patients. At the multivariate analysis for OS, normal albumin level (HR 0.68, 95% CI 0.57–0.81, p < 0.0001), CEA levels within normal ranges (HR 0.68, 95% CI 0.57–0.82, p < 0.0001), NLR < 3 (HR 0.62, 95% CI 0.52–0.74, p < 0.0001), ECOG PS 0 (HR 0.51, 95% CI 0.42–0.61, p < 0.0001), and prior surgery (HR 0.80, 95% CI 0.65–0.99, p = 0.036) were positive prognostic factors. Conclusion The updated findings, derived from an expanded cohort, further support the adoption of durvalumab in combination with gemcitabine and cisplatin in routine clinical practice, reinforcing the efficacy and safety outcomes demonstrated in the phase III TOPAZ‐1 trial.