Citations

Abstract Description Weakened germinal center responses by the aged immune system result in diminished immunity against pathogens and reduced efficacy of vaccines. Prolonged contacts between activated B cells and CD4+ T cells are crucial to germinal center formation and Tfh differentiation, but it is unclear how aging impacts the quality of this interaction. Peptide immunization confirmed that aged mice have decreased expansion of antigen-specific GC B cells and reduced antibody titers. Aging was associated with accumulated Tfh cells with reduced BCL6 and increased CD39. When activated in vitro by aged antigen-presenting cells, young CD4+ naïve T cells generated reduced numbers of activated cells with upregulated CD40L. To determine the contribution of cell-extrinsic influences on antigen-specific Tfh induction, young, antigen-specific B and CD4+ T cells were transferred into aged hosts prior to immunization. Transferred cells had reduced expansion and differentiation into germinal center B cell and Tfh and reduced antigen-specific antibody titers when compared to young hosts. Young CD4+ T cells transferred into aged hosts differentiated into Tfh cells with reduced PD-1 and BCL6, and increased CD39. Recent studies have focused on immune profiling of CD4 T cells in young and aged mice following immunization. These results highlight the role of the lymphoid microenvironment in modulating CD4+ T cell differentiation, which contributes to impaired initiation and maintenance of germinal centers. Funding Sources Research was supported by NIH/NIA R01 AG080037 and the Mayo Clinic Robert and Arlene Kogod Center on Aging Innovation Award UL1R002377 to J.L. Topic Categories Vaccines and Immunotherapy (VAC)

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 CAR-T cell therapy has shown remarkable success in treating hematologic cancers; however, its efficacy in solid tumors remains limited, partly due to tumor heterogeneity, immune suppression within the tumor microenvironment, and inefficient CAR-T cell infiltration. I hypothesize that increasing the sensitivity of tumor cells to cytokine-mediated killing could enhance the therapeutic impact of CAR-T cells in solid tumors. I propose that targeting Adenosine Deaminases Acting on RNA 1 (ADAR1), an enzyme that edits double-stranded RNA (dsRNA) to prevent immune activation, may be one such strategy. ADAR1 balances inflammation by preventing the overactivation of dsRNA sensors such as PKR, MDA5, and RIG-I. However, in the absence of ADAR1, accumulating dsRNA structures will trigger these dsRNA sensors and alter the tumor cell intrinsic response to T cell-derived cytokines, such as IFNγ and TNF. My data provide evidence that inhibition of ADAR1 in tumor cells sensitize them to killing by cytokines, but it remains unclear how signaling from dsRNA sensors crosstalk with those from cytokine receptors to regulate cell survival or death. My study aims to elucidate the interplay between these different signaling pathways in the absence of ADAR1, and how this interplay determines the efficacy of CAR-T cells against solid tumors. My experimental observations could provide insights into novel therapeutic strategies to improve CAR-T cell potency against resistant tumor types. Funding Sources Mayo Clinic Cancer Center Fellowship Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Description PBC is an archetypal autoimmune disease where the dominant autoantigen is mitochondrial PDC-E2. Hitherto, PBC treatment has been neither specific nor directed toward restoration of tolerance. CNP-104, a tolerogenic nanoparticle encapsulating PDC-E2, is hypothesized to restore tolerance to pathogenic T cells. We conducted a Phase 2a First-in-Human randomized controlled trial of CNP-104 in PBC patients unresponsive/intolerant to ursodeoxycholic acid. The 42 enrolled subjects were randomized 2:1 to receive two doses of CNP-104 or placebo (PBO) one week apart and were followed for 120 days to assess safety and treatment durability. CNP-104 was safe and well-tolerated. Compared to PBO, the CNP-104 group had a reduced percentage of antigen-specific Th17 T cells and increased antigen-specific tolerogenic CD8+ T cell counts (TIGIT, GARP). ALP, ALT, and total bilirubin were similar between groups. Clinically relevant effects on albumin levels, liver stiffness, and the rate of change in 5-year UK-PBC risk for liver outcomes were improved in the CNP-104 group compared to PBO. This trial is limited by small size and data variability. Induction of antigen-specific T cell tolerance, shown by reduced Th17 cells and increased TIGIT and GARP cells correlates with improved liver stiffness, albumin, and UK-PBC. These findings suggest the CNP-104 tolerogenic mechanism of action translates to clinical benefits. These data support future studies with this unique therapeutic platform to treat PBC. Topic Categories Therapeutic Approaches to Autoimmunity (THER)

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 A durable T cell immunity against cancer is dependent on a continually refilling of effector CD8+ T cells. Thymic output has been correlated with favorable prognosis in cancer patients of advanced ages, suggesting thymus is an important source for refilling T cells that are capable of controlling cancer progression in the periphery. However, it remains unclear how PD-1 signals regulate the cytotoxic potential of CD8+ T cells in thymus before they egress to the periphery to establish durable anti-tumor immunity. Here we report PD-1 signaling prelimits thymic single positive (SP) CD8+ T cells to acquire cytotoxic potential by downregulating Gzmb and Nkg7, which are required for CD8+ T cell-mediated cytotoxicity. Unexpectedly, we found the absence of Pdcd1 also led to upregulation of Tox and Tim3 in thymic SP CD8+ T cells, thereby pre-programming these cells for exhaustion within the tumor. Thus, the absence of PD-1 signals in CD8+ T cells only partially delayed tumor growth in peripheral tissues. To improve therapeutic effects of PD-1 inhibition, we observed that tumor growth in CD8-specific Pdcd1 conditional knockout mice was further suppressed by treatment with a PD-L1 antibody that blocks PD-L1 and CD80 interaction. Our results imply that PD-1 inhibition promotes the expansion of effector cytotoxic CD8+ T cells but also retains T cell exhaustion potential, which can be mitigated by PD-L1 blockade that allows more CD80 to provide costimulatory signals to T cells via CD28. Funding Sources NCI R01CA256927 NCI R01CA261932 NCI P30CA15083 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Description Immunotherapy combinations can improve patient outcomes, yet the interactions within the tumor microenvironment (TME) that drive therapeutic synergy are poorly understood. Tumor establishment drives monocyte recruitment and differentiation into tumor-associated macrophages (TAMs), which have essential roles in coordinating immune responses and are thus attractive targets for therapeutic modulation. In a murine model of combination anti-PD-1 and anti-PD-L1 checkpoint blockade, tumor control was associated with increased infiltration of CD8+ T cells and M1-like repolarization of TAMs. Live-cell imaging of the tumor microenvironment revealed stable contacts between tumor-infiltrating CD8+ T cells and TAMs, in which the extent of the contact interfaces increased with combination immunotherapy. Treatment with anti-PD-L1 was able to increase macrophage expression of pro-inflammatory factors and phagocytic activity, suggesting a role for TAMs to reactivate CD8+ T cells in the TME. However, co-treatment with anti-PD-1 was ultimately necessary for tumor control, indicating the need for combination targeting of the TME. Funding Sources Mayo Clinic David and Margaret Grohne Cancer Immunotherapy Pilot Award Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

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 Hypertensive disorders of pregnancy (HDP) include preeclampsia (PE) and gestational hypertension (gHTN), both suspected to be immune-driven pathologies. Our data shows that both PE and gHTN involve systemic immune cell activation and increased proinflammatory cytokines however it is unclear how these immune profiles play a role in the pathogenesis of disease. We hypothesize that pregnancies complicated with HDP have dysregulated immune profiles, including hyperactive T cells which are activating the vascular endothelium. We have investigated the circulating immune profiles of patients with normotensive (NT), PE or gHTN pregnancies at the RNA, protein and cellular levels, which show significant alterations in T cells, among others. We have seen increased levels of IL-6, CXCL9 and CXCL10 in the plasma of HDP patients vs NT, suggesting the polarization and recruitment of T cells. Using cytometry by time of flight (CyTOF), we have seen changes in expression of CD11a and CXCR3 on CD4+, CD8+ and yd T cells in HDP compared to NT. Single cell RNA sequencing of CD4+ and CD8+ T cells from PE vs NT shows increased expression of genes related to pathways such as allograft rejection, angiogenesis and cytokine signaling. Our in vitro co-culture experiments examine the ability of immune cells, specifically T cells, to activate endothelial cells. These studies will address the functional contribution of T cells to endothelial activation, which is a hallmark of the hypertension seen in HDP. Topic Categories Cellular Adhesion, Migration, and Inflammation (CAM)

Abstract Description Preeclampsia (PE) is a complication of pregnancy characterized by de novo hypertension after 20 weeks of gestation with end-organ damage. Even though symptoms of PE rapidly resolve after delivery, patients have higher incidence of cardiovascular diseases in the years postpartum. Immunological changes have been observed in PE but it is unknown if these are sustained in the postpartum period. Our goal was to address the immune changes in the first year postpartum in PE as compared to normotensive (NT) as well as nulliparous women. We saw that both PE and NT pregnancies had altered immune system, especially decreased CD14+ monocytes and increased circulating pro-inflammatory cytokines when compared to nulliparous patients even one year after delivery. Furthermore, our intracellular cytokine staining analysis following stimulation suggest altered response in the postpartum period in PE with a predominant anti-inflammatory response vs NT. Overall this work suggest that the immune system remains different from nulliparous women at 1 year postpartum, even in cases of normotensive pregnancies, and is further altered following PE-complicated pregnancies. Although further analysis is ongoing, this shows that the immune system does not go back to pre-pregnancy levels in the postpartum period and that changes induced by pregnancy are at least in part sustained. We will further expand this work to address the functional changes in immune cells and the mechanisms underlying these changes. Topic Categories Mucosal and Regional Immunology (MUC)