O046 / #556 Topic:AS02 - Animal Models ABSTRACT CONCURRENT SESSION 07: COGNITION IMPAIRMENT IN SLE — RECENT ADVANCEMENT AND EMERGING RESEARCH 23-05-2025 1:40 PM - 2:40 PM Unclear mechanisms underlying diffuse NPSLE (psychosis, anxiety disorder, cognitive dysfunction) may lead to the devastating impact of this disease on patients’ health-related quality of life, representing a major unmet need in the field. In prior work, systemic depletion of CD4+ T cells ameliorated both systemic disease and behavior deficits in SLE- and NPSLE-prone MRL/lpr mice by reducing choroid plexus-infiltrating CD4+ T cells as well as indirectly preventing CD8+ T cell infiltration. Further, increased markers of exhaustion were identified in choroid plexus-infiltrating CD8+ T cells of MRL/lpr mice. However, NPSLE is heterogeneous in its presentation; choroid plexus infiltrate is not a fully penetrant hallmark of human NPSLE or other disease models. Thus, it is critical to elucidate the contribution of T cell subsets outside of the choroid plexus to NPSLE. Perfused brains of female SLE- and NPSLE-prone CReCOM (8 mo old; n=4-11) and B6.Sle1Sle2Sle3(TC; 2 and 8 mo old; n=3-4) mice, and respective control strains, were extracted after intravenous CD45 labeling to exclude remaining circulating immune cells, dural meninges were removed, and cells were analyzed by flow cytometry. Young female CD45.1 (Jackson 033076) and TC mice were used to generate reciprocal head-shielded BM chimeric mice with busulfan treatment to clear remaining BM (CD45.1 BM::CD45.1; TC BM::CD45.1; CD45.1 BM::TC; TC BM::TC). Mice underwent behavioral tasks 10 weeks post-transfer. Live CD45+cells were FACSorted from pooled cell suspensions (n=3/group to account for biological variability) for cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq; 10X Genomics 3’ v3.1). Data from CD45.1 BM::CD45.1 and TC BM::TC chimeras were analyzed in R using the Seurat package. Post-filtering, ~10K cells/chimera were maintained. Extravascular CD8+ T cells are significantly increased in the brains of CReCOM and TC mice (8 mo old) compared to their respective control strains. Further, this increase is evident at 2 months of age in TC mice. Evaluation of extravascular T cells by CITE-seq in CD45.1 BM::CD45.1 and TC BM::TC chimeric mice was carried out to mimic that of control and NPSLE-prone strains, respectively, in systemic disease and behavioral phenotypes. Similar to data from the kidney and choroid plexus of MRL/lpr mice, we show expansion of an exhausted (Pdcd1, Eomes, Tox) CD8+ T cell subset. We also identify an expanded IL-17-producing gamma-delta T cell subset previously unassociated with NPSLE-like disease but implicated in neuro-autoimmune and -degenerative diseases (multiple sclerosis, Alzheimer’s disease). As CReCOM and TC mice do not present with the characteristic choroid plexus infiltrate of the MRL/lpr strain, these subsets mediate their activity in the brain parenchyma or nondural meninges. We find expansion of an exhausted CD8+ T cell subset in the brain of NPSLE-prone mice that may transition from a highly activated T cell state. An IL-17-producing gamma-delta T cell subset not previously associated with NPSLE was also expanded. Future studies will interrogate these specialized T cell subsets for their location of action to further uncover their role of in NPSLE-like disease.

Background T cells play a significant role in the pathogenesis and end-organ damage observed in systemic lupus erythematosus (SLE). Previous studies using murine SLE models have demonstrated that kidney infiltrating T cells (KITs) differ substantially from their peripheral counterparts. Notably, KITs exhibit greater heterogeneity, characterized by a prominent population of resident memory and exhausted/dysfunctional T cells, along with a transitional or early exhausted population. In this study, our aim was to investigate T cell heterogeneity in multiple organs affected by SLE. We hypothesized that T cells isolated from different target organs would display distinct functional and transcriptional programs influenced by tissue-specific reprogramming. Additionally, we expected isolated clonal expansion in each target organ, with less clonal expansion in the spleen. Methods Using the MRL. Faslpr model, which recapitulates numerous features human SLE, we isolated CD4 and CD8 T cells from five organs commonly impacted in SLE: kidney, liver, lung, spleen, and skin. T cells were obtained from female mice (18 wks or older) and male mice (26 wks or older). Single cell suspensions were prepared through chemical and mechanical dissociation. High-dimensional flow cytometry, metabolic analysis, functional studies, and scRNA-seq and TCR-seq were performed to analyze T cell heterogeneity. Results Our findings revealed substantial variability in T cell function, metabolism, and phenotype across the different organs examined. Consistent with previous observations, the spleen was comprised of mainly effector T cells. Similar findings were observed in mucosal tissue associated infiltrating T cells isolated from lung and skin, which exhibited increased cytokine production, glucose uptake, and enhanced mitochondrial membrane potential. Conversely, T cells infiltrating solid organs such as the kidney and liver exhibited a more suppressed/exhausted phenotype, characterized by expression of exhaustion markers, an altered metabolic profile, and specific transcriptional program. Interestingly, clonal expansion was not organ-specific, as expanded clones were observed in all target organs. Conclusion Our data underscore the influence of tissue parenchyma on the phenotype and transcriptional program of infiltrating immune cells. Specifically, solid organs (liver and kidney) possess the capacity to suppress T cell responses, potentially limiting self-damage. In contrast, T cells from lymphoid and mucosal tissues (skin and lung) exhibit a more activated phenotype. This dichotomy can be explained by the evolutionary need to minimize immune-mediated damage in solid organs while maintaining effective immunity against external infections in mucosal tissues. By gaining a deeper understanding of how immune cells are modulated within distinct tissue microenvironments, we may identify strategies to selectively target organ-specific manifestations while minimizing systemic immunosuppression.

Nucleic acid-specific Toll-like receptors (TLRs) have been implicated in promoting disease pathogenesis in systemic lupus erythematosus (SLE). Whether such TLRs mediate disease onset, progression, or both remains undefined; yet the answer to this question has important therapeutic implications. MyD88 is an essential adaptor that acts downstream of IL-1 family receptors and most TLRs. Both global and B cell-specific Myd88 deficiency ameliorated disease in lupus-prone mice when constitutively deleted. To address whether Myd88 was needed to sustain ongoing disease, we induced B cell-specific deletion of Myd88 after disease onset in MRL.Faslpr mice using an inducible Cre recombinase. B cell-specific deletion of Myd88 starting after disease onset resulted in ameliorated glomerulonephritis and interstitial inflammation. Additionally, treated mice had reduced autoantibody formation and an altered B cell compartment with reduced ABC and plasmablast numbers. These experiments demonstrate the role of MyD88 in B cells to sustain disease in murine lupus. Therefore, targeting MyD88 or its upstream activators may be a viable therapeutic option in SLE.

The endosomal Toll-like receptor 7 (TLR7) is a major driver of murine and human systemic lupus erythematosus (SLE). The role of TLR7 in lupus pathogenesis is enhanced when the regulatory role of TLR9 is absent. TLR7 signaling in plasmacytoid DCs (pDC) is generally thought to be a major driver of the IFN response and disease pathology; however, the cell types in which TLR7 acts to mediate disease have not been distinguished. To address this, we selectively deleted TLR7 in either CD11c+ cells or CD19+ cells; using a TLR7-floxed allele, we created on the lupus-prone MRL/lpr background, along with a BM chimera strategy. Unexpectedly, TLR7 deficiency in CD11c+ cells had no impact on disease, while TLR7 deficiency in CD19+ B cells yielded mild suppression of proteinuria and a trend toward reduced glomerular disease. However, in TLR9-deficient MRL/lpr mice with accelerated SLE, B cell-specific TLR7 deficiency greatly improved disease. These results support revision of the mechanism by which TLR7 drives lupus and highlight a cis regulatory interaction between the protective TLR9 and the pathogenic TLR7 within the B cell compartment. They suggest B cell-directed, dual TLR7 antagonism/TLR9 agonism or dual TLR7/9 antagonism as a potential future therapeutic strategy to treat SLE.

Background Lupus nephritis is the most common life-threatening end-organ complication of SLE. Interstitial infiltrates, specifically T cells, are major predictors of disease outcomes. We recently determined that kidney-infiltrating T cells (KITs) are suppressed after kidney infiltration and exhibit an exhausted phenotype. Notably, the kidneys of nephritic lupus-prone (MRL.Faslpr) mice upregulate PD-L1, which we hypothesize is one mechanism inducing KIT suppression. IFNγ is the major inducer of PD-L1 and given the known role of IFNγ in lupus nephritis, we postulated that IFNγ induces a protective program in the kidneys of lupus-prone mice, in direct contrast to the proinflammatory role of IFNγ in the hematopoietic compartment. Methods MRL. Faslpr mice develop autoantibodies, proteinuria, dermatitis, and glomerulonephritis. Others have previously shown that global IFNγ receptor deficiency (IFNγR-/-) ameliorates disease in this mouse model. To determine if IFNγ signaling on parenchymal cells regulates disease, we generated bone marrow chimeras by transferring congenically labeled WT immune cells into either wild-type (WT) or IFNγR-/- MRL.Faslpr recipients. If our hypothesis is correct, then the IFNγR-/- recipients would have more severe disease than their WT counterparts. Chimerization occurred at 4-6 weeks of age and female and male mice were analyzed for disease pathology at 23 and 27 weeks post-chimerization respectively. Analysis included proteinuria, renal histology for both interstitial and glomerular disease, dermatitis, autoantibody production, and immune cell activation. Survival analysis was performed on female mice. Additional analysis focused specifically on T cell phenotypes. Results IFNγR-/- MRL.Faslpr recipient mice exhibited more severe and rapid disease onset than WT recipient controls. While proteinuria was not different between the two groups, the IFNγR-/- recipients had more severe glomerulonephritis (p< 0.005) and interstitial disease (p< 0.001). Consistent with these findings, IFNγR-/- recipients had reduced survival (p< 0.05). As expected, IFNγR deficiency resulted in reduced PD-L1 expression. When examining infiltrates, KITs isolated from IFNγR-/- recipients exhibited increased expression of Tim3 and PD-1. Conclusions These experiments suggest that parenchymal IFNγR signaling results in upregulation of protective mechanisms which reduce kidney disease and alter T cell phenotypes. This contrasts with global IFNγR-/- which ameliorated kidney disease. Overall, this finding argues that suppression of IFNγ, and possible other inflammatory mediators, may have differential effects on specific cell lineages and that global suppression of IFNγR may have both positive and negative effects on disease pathogenesis. This will need to be considered when devising targeted therapies. Acknowledgments This work was supported by the Lupus Research Alliance (Novel Research Grant), NIAID (R01 AI137132), and J.T was supported by NIAMS 5K08AR075056.

Background/Purpose Lupus nephritis is the most common life-threatening end-organ complication of SLE. Interstitial infiltrates, specifically T cells, are major predictors of disease outcomes. We recently determined that kidney-infiltrating T cells (KITs) are suppressed after kidney infiltration and exhibit an exhausted phenotype. We hypothesize that one mechanism of suppression is an IFNγ inducible immunosuppressive on the tissue parenchyma. Previously we and others have shown that PD-L1 is upregulated on the parenchyma of lupus nephritis patients and lupus-prone mice. KITs have high expression of PD1 when compared to lymphoid T cells from the same mouse. Furthermore, IFNγ is the major inducer of PD-L1. Therefore, we postulated that IFNγ induces a protective program mediated by PD-L1 which results in suppresses immune destruction of the kidney in lupus nephritis. Methods MRL Faslpr mice develop autoantibodies, proteinuria, dermatitis, and glomerulonephritis. To determine if PD-L1 and IFNγ signaling on parenchymal cells regulates disease, we generated bone marrow chimeras by transferring congenically labeled WT immune cells into either wild-type (WT) or IFNγR-/- MRL.Faslpr or PD-L1-/- MRL.Faslpr recipients. If our hypothesis is correct, then both IFNγR-/- and PD-L1-/- recipients would have more severe disease than their WT counterparts. Chimerization occurred at 4-6 weeks of age, female and male mice were analyzed for disease pathology at 4-6 months post-chimerization depending on genetic background. Analysis included proteinuria, renal histology for both interstitial and glomerular disease, dermatitis, autoantibody production, and immune cell activation. Additional analysis focused specifically on T cell phenotypes. Results As hypothesized, the IFNγR-/- MRL.Faslpr recipient mice exhibited more severe and rapid disease onset than WT recipient controls. The IFNγR-/- recipients had more severe glomerulonephritis (p< 0.005) and interstitial disease (p< 0.001). Consistent with these findings, IFNγR-/- recipients had reduced survival (p< 0.05). As expected, IFNγR deficiency resulted in reduced PD-L1 expression. However, there was no appreciable difference between PD-L1-/- MRL.Faslpr recipient compared to controls, they had no increase in mortality nor significant difference is nephritis status. When examining infiltrates, KITs isolated from IFNγR-/- recipients exhibited increased expression of Tim3 and PD-1. Similar to the disease endpoints, these T cells changes were not noted in the PD-L1-/- MRL.Faslpr recipient mice . Conclusion These experiments suggest that parenchymal IFNγR signaling results in upregulation of protective mechanisms which reduce kidney disease and alter T cell phenotypes. While we hypothesized that this would be mediated through IFNγ induced PD-L1 the latter hypothesis does not appear to be supported. This data contrasts with global IFNγR-/- which ameliorated kidney disease and global PD-L1 deficiency which led to increased mortality in MRL.Faslpr mice. Overall, suppression of IFNγ, and possible other inflammatory mediators, may have differential effects on specific cell lineages and that global suppression of IFNγR may have both positive and negative effects on disease pathogenesis. Furthermore, this work shows that this IFNγ response is independent of PD-L1 signaling. In all, these findings should be considered when devising novel targeted therapies.