Background and Objectives : Oral cladribine tablet (CladT) therapy is efficacious for relapsing multiple sclerosis (MS). However, the mechanisms by which cladribine exerts benefit in MS remain unclear, particularly regarding its effects on compartmentalized inflammation within the CSF. Methods : Transcriptional profiles along with T and B lymphocyte receptor repertoires from CSF and blood were obtained by single-cell sequencing methods from a single site participating in a phase IV clinical trial investigating the impact of cladribine treatment for MS. Blood and CSF samples from patients were obtained immediately before starting CladT therapy, and they were randomized to also provide additional samples at either 5 weeks, 10 weeks, 1 year, or 2 years after CladT therapy. Thirty-four samples from 13 individuals with relapsing MS before and after treatment were available to test the hypothesis that CladT alters the composition and phenotype of lymphocytes in the CSF, including paired baseline and post-CladT CSF samples obtained from 4 unique participants. Results : We found that treatment with CladT profoundly altered cellular composition, but not the transcriptional phenotype, of immune cells in the CSF. In particular, we identified a reduction in switched memory B cells but recovery of naive B cells in the CSF, similar to our findings in blood. In addition, populations of CD4 Treg cells emerged early after CladT therapy and remained elevated 1 year later in the CSF, but not in the blood. Antigen receptor sequencing revealed a moderate decrease in numbers of large clonally expanded CD8 T cell clones (>10 cells/clone) primarily in the CSF, but also in the blood after CladT treatment. Discussion : Our results identified unique cellular dynamics and changes in T cell and B cell clonality in both tissues, which can potentially explain long-term beneficial effects of CladT therapy in MS, including preservation of immune function and a relatively low number of side effects. Altogether, this study demonstrates that CladT treatment had a substantial impact not only on blood but also on the CSF compartment, highlighting the importance of cross-tissue analysis for better understanding of effect and the mechanism of action of disease-modifying therapies.

Top 5 gene ontology pathways enriched in each cell type upon chemoradiation related to Figure 3D

Human leukocyte antigen (HLA) alloimmunization poses a significant challenge in various clinical settings, including platelet refractoriness and allogeneic solid-organ and stem cell transplants. We have explored HLA-Fc fusion proteins as a novel therapeutic strategy for suppressing or reversing unwanted HLA alloimmunization. These fusion proteins, combining HLA antigen specificity with Fc-mediated cytotoxicity, show promise in targeting specific antibody-producing B cells. Initial studies using B cell hybridomas demonstrate the feasibility and selectivity of HLA-Fc in killing target cells of cognate specificities. The protocols in this chapter outline the design, production, and evaluation of HLA-Fc proteins, emphasizing their efficacy in vitro and in immunodeficient mouse models. Further research is needed to validate these findings in humanized murine alloimmunization models, potentially providing new therapeutic approaches for managing HLA-sensitized patients in clinical practice.

A key feature of the intestinal immune system is balancing pathogen defense with antigen-specific tolerance to commensal bacteria. Here, using conditional deletion models, we identified the transcription factor BHLHE40 as a central regulator of group 3 innate lymphoid cell (ILC3)- and RORγt⁺ antigen-presenting cell (APC)-dependent mucosal immunity. In ILC3s, BHLHE40 drove transcription of cytokine effector programs and maintenance of mucosal immunity. Cytokine TL1A stimulation and inflammation induced Bhlhe40 expression, amplifying these programs through epigenetic modulation of chromatin accessibility at effector loci. Bhlhe40 was also highly expressed in RORγt⁺ APCs, where it was required for the generation of antigen-specific Tregs. In parallel, BHLHE40 integrated microbial cues to promote expression of the co-stimulatory molecule OX40L by ILC3s, further promoting antigen-specific Treg induction. Together, these findings define Bhlhe40 as a coordinated regulator of barrier immunity and support a model in which ILC3s and RORγt⁺ APCs act in concert to shape antigen-specific intestinal immunity.

Cytoscape node information related to Fig. 1l

Abstract Despite advances in screening and prevention, cervical cancer remains a leading cause of cancer-related deaths worldwide, underscoring the need for better treatments. In this study, we conducted a multicohort longitudinal study of human cervical tumors and the tumor microenvironment during chemoradiotherapy (CRT) and integrated RNA sequencing and single-cell transcriptomics to define the cellular and molecular programs shaping cell interactions and how CRT alters them. The analysis identified multiple therapeutic targets in CRT-resistant tumors, notably including MDM2, a key mediator of radiation responses in tumor and immune cells. MDM2 inhibition enhanced the effects of radiotherapy in human papillomavirus (HPV)–positive, TP53 wild-type cervical cancer cells; improved radiation response; and reshaped the immune landscape in preclinical models. These findings highlight the potential of combining MDM2 inhibition with CRT to overcome resistance and improve patient outcomes. The insights into therapy-induced changes in tumor and immune compartments could guide improved strategies against treatment-resistant HPV-positive cancers. Significance: Mapping of the impact of chemoradiation on cellular interactions in cervical cancer reveals how treatment reshapes the tumor microenvironment and highlights targets for developing future immunotherapeutic approaches. See related commentary by Klopp, p. 1540

Singe-cell expression matrix for custom clustering annotation related to Figure 2A

Exposure to particles is a driver of several inflammatory diseases. Here, we investigated macrophage responses to monosodium urate crystals, calcium pyrophosphate crystals, aluminum salts, and silica nanoparticles. While each particle induced a distinct gene expression pattern, we identified a common inflammatory signature and acute activation of lysosomal acidification genes. Using monosodium urate crystals as a model, we demonstrated that this lysosomal gene program is regulated by a 5'-prime-AMP-activated protein kinase (AMPK)-dependent transcriptional network, including TFEB, TFE3, and the epigenetic regulators DNA methyl transferase 3a (DNMT3A) and DOT1L. This lysosomal acidification program operates in parallel with, but largely independently of, a JNK-AP-1-dependent network driving crystal-induced chemokine and cytokine expression. These findings reveal a bifurcation in pathways governing inflammatory and lysosomal responses, offering insights for treating particle-associated diseases.

Single-cell transcriptomics applied to cerebrospinal fluid (CSF) for elucidating the pathophysiology of neurologic diseases has produced only a preliminary characterization of CSF immune cells. CSF derives from and borders central nervous system (CNS) tissue, allowing for comprehensive accounting of cell types along with their relative abundance and immunologic profiles relevant to CNS diseases. Using integration techniques applied to publicly available datasets in combination with our own studies, we generated a compendium with 139 subjects encompassing 135 CSF and 58 blood samples. Healthy subjects and individuals across a wide range of diseases, such as multiple sclerosis (MS), Alzheimer's disease, Parkinson's disease, COVID-19, and autoimmune encephalitis, were included. We found differences in lymphocyte and myeloid subset frequencies across different diseases as well as in their distribution between blood and CSF. We identified what we believe to be a new subset of AREG+ dendritic cells exclusive to the CSF that was more abundant in subjects with MS compared with healthy controls. Finally, transcriptional cell states in CSF microglia-like cells and lymphoid subsets were elucidated. Altogether, we have created a reference compendium for single-cell transcriptional profiling encompassing CSF immune cells useful to the scientific community for future studies on neurologic diseases.

The transcriptional programs that enable CD4 and CD8 T cells to mediate effective anti-tumor immunity remain incompletely defined. Here, we identify distinct, therapy-specific roles for the transcriptional regulator Bhlhe40 in CD4 and CD8 T cells, revealing divergent requirements during anti-PD-1 versus anti-CTLA-4 immune checkpoint therapy (ICT). Using conditional knockout mice, we show that anti-PD-1 efficacy depends on CD8 T cell-intrinsic Bhlhe40 and may also require its expression in CD4 T cells, whereas anti-CTLA-4 relies primarily on CD4 T cell-intrinsic Bhlhe40 and remains effective without Bhlhe40 in CD8 T cells. Loss of Bhlhe40 skews CD8 T cells toward TCF-1-expressing naïve and progenitor exhausted-like states, particularly in the absence of ICT. Bhlhe40 sustains CD8 effector and exhausted phenotypes, promotes IFN-γ production, and supports glycolytic and mitochondrial programs, with Bhlhe40 deficiency leading to impaired glycolysis under either anti-PD-1 or anti-CTLA-4 ICT, and reduced mitochondrial function primarily during anti-PD-1. In addition, CD8 T cell-intrinsic Bhlhe40 is required for full ICT-induced remodeling of the tumor myeloid compartment from CX3CR1+ macrophages to iNOS+ macrophages. In contrast, anti-CTLA-4 can drive tumor rejection and partial macrophage remodeling in the absence of CD8 T cell-intrinsic Bhlhe40, provided CD4 T cell function is intact. Analysis of human cancer datasets revealed that BHLHE40 is enriched in tumor-reactive and activated/exhausted CD8 T cells, where its expression is inversely correlated with TCF7 (TCF-1) and positively associated with TOX and IFNG. Moreover, subsets of CD8 T cells expressed higher levels of BHLHE40 in basal and squamous cell carcinoma responders compared with non-responders. Together, these findings establish Bhlhe40 not only as a transcriptional coordinator of T cell effector programs and metabolic fitness, but also as a therapy-specific, subset-dependent determinant of ICT efficacy, providing a mechanistic basis for the divergent modes of action of anti-PD-1 versus anti-CTLA-4.

Approximately 1.5 billion people are infected with parasitic worms. The dynamics of priming, expansion, differentiation, contraction, and memory responses by helminth-specific Th2 cells remain understudied. We aim to create a CD4+ TCR transgenic mouse specific for H. bakeri, a gastrointestinal nematode, that will enable us to track antigen-specific CD4+ T cells. For this purpose, we have established H. bakeri infection in mice with a fixed TCR beta chain, termed TcliB. CD4+ T cells from TcliB mice largely use this transgenic TCR beta chain paired with a diversity of TCR alpha chains. We sorted small intestine lamina propria CD4+ T cells from both uninfected and H. bakeri-infected TcliB mice for scRNA-sequencing to determine their gene expression and TCR alpha chain usage. We observed heterogeneity among lamina propria CD4+ T cells subtypes, including several clusters of Th2 cells whose relative abundance varied upon helminth infection. TCR alpha chain sequencing identified expanded T cell clones in infected mice, with specific TCRs present in multiple mice. We are testing these TCRs for reactivity against H. bakeri antigens and will examine whether T cells expressing these TCRs expand in vivo upon infection. Insights from this project will provide novel perspectives into how a particular helminth-specific CD4+ T cell clone may take multiple polarization fates during infection, and whether T cell responses change during co-infection with other intestinal microbes. NIH R01AI162918 Microbial, Parasitic, and Fungal Immunology (MPF)

Disease tolerance is a host response to infection that limits collateral damage to host tissues while having a neutral effect on pathogen fitness. Previously, we found that the pathogenic lactic acid bacterium Streptococcus pyogenes manipulates disease tolerance using its aerobic mixed-acid fermentation pathway via the enzyme pyruvate dehydrogenase, but the microbe-derived molecules that mediate communication with the host's disease tolerance pathways remain elusive. Here we show in a murine model that aerobic mixed-acid fermentation inhibits the accumulation of inflammatory cells including neutrophils and macrophages, reduces the immunosuppressive cytokine interleukin-10, and delays bacterial clearance and wound healing. In infected macrophages, the aerobic mixed-acid fermentation end-products acetate and formate from streptococcal upregulate host acetyl-CoA metabolism and reduce interleukin-10 expression. Inhibiting aerobic mixed-acid fermentation using a bacterial-specific pyruvate dehydrogenase inhibitor reduces tissue damage during murine infection, correlating with increased interleukin-10 expression. Our results thus suggest that reprogramming carbon flow provides a therapeutic strategy to mitigate tissue damage during infection.

Diverse human leukocyte antigens (HLA) play a crucial role in adaptive immune responses via peptide presentation. Unrelated to this role, alloimmunization to mismatched HLA represents a significant barrier to allogeneic transplantation. Despite the complexity of HLA structures and the diversity of alleles, advancements in recombinant HLA protein production have enabled their use in diagnostics and are paving the way for therapeutic applications. HLA-Fc fusion proteins, combining the extracellular domains of HLA molecules with the Fc region of immunoglobulins, offer a promising approach for antigen-specific immunotherapy. This review examines the engineering challenges and therapeutic potential of HLA-Fc fusion proteins in mitigating unwanted HLA alloimmunization and antibody-mediated rejection (AMR) following transplantation. HLA-Fc fusion proteins provide enhanced stability and effector functions, enabling the selective targeting and depletion of anti-HLA antibody-producing cells. Preclinical studies demonstrate their efficacy in neutralizing anti-HLA antibodies and depleting cognate antibody-producing cells, highlighting their potential to improve transplant outcomes and reduce AMR. Future research may focus on optimizing these fusion proteins, understanding their effects on primary B cells and long-lived plasma cells, and exploring combination therapies to enhance their clinical efficacy.