Single-cell transcriptomics unveil profiles and interplay of immune subsets in rare autoimmune childhood Sjögren's disease.

Childhood Sjögren's disease represents critically unmet medical needs due to a complete lack of immunological and molecular characterizations. This study presents key immune cell subsets and their interactions in the periphery in childhood Sjögren's disease. Here we show that single-cell RNA sequencing identifies the subsets of IFN gene-enriched monocytes, CD4+ T effector memory, and XCL1+ NK cells as potential key players in childhood Sjögren's disease, and especially in those with recurrent parotitis, which is the chief symptom prompting clinical visits from young children. A unique cluster of monocytes with type I and II IFN-related genes is identified in childhood Sjögren's disease, compared to the age-matched control. In vitro regulatory T cell functional assay demonstrates intact functionality in childhood Sjögren's disease in contrast to reduced suppression in adult Sjögren's disease. Mapping this transcriptomic landscape and interplay of immune cell subsets will expedite the understanding of childhood Sjögren's disease pathogenesis and set the foundation for precision medicine.

Placental circulating T cells: a novel, allogeneic CAR-T cell platform with preserved T-cell stemness, more favorable cytokine profile, and durable efficacy compared to adult PBMC-derived CAR-T.

BACKGROUND: Chimeric antigen receptor (CAR)-T cell quality and stemness are associated with responsiveness, durability, and memory formation, which benefit clinical responses. Autologous T cell starting material across patients with cancer is variable and CAR-T expansion or potency can fail during manufacture. Thus, strategies to develop allogeneic CAR-T platforms including the identification and expansion of T cell subpopulations that correspond with CAR-T potency are an active area of investigation. Here, we compared CAR-T cells generated from healthy adult peripheral blood T cells versus placental circulating T (P-T) cells. METHODS: CAR-T cells from healthy adult peripheral blood mononuclear cells (PBMCs) and P-T cells were generated using the same protocol. CAR-T cells were characterized in detail by a combination of multiparameter flow cytometry, functional assays, and RNA sequencing. In vivo antitumor efficacy and persistence of CAR-T cells were evaluated in a Daudi lymphoma xenograft model. RESULTS: P-T cells possess stemness advantages compared with T cells from adult PBMCs. P-T cells are uniformly naïve prior to culture initiation, maintain longer telomeres, resist immune checkpoint upregulation, and resist further differentiation compared with PBMC T cells during CD19 CAR-T manufacture. P-T CD19 CAR-T cells are equally cytotoxic as PBMC-CD19 CAR-T cells but produce less interferon gamma in response to lymphoma. Transcriptome analysis shows P-T CD19 CAR-T cells retain a stem-like gene signature, strongly associate with naïve T cells, an early memory phenotype, and a unique CD4 T cell signature compared with PBMC-CD19 CAR-T cells, which enrich for exhaustion and stimulated memory T cell signatures. Consistent with functional data, P-T CD19 CAR-T cells exhibit attenuated inflammatory cytokine and chemokine gene signatures. In a murine in vivo model, P-T CD19 CAR-T cells eliminate lymphoma beyond 90 days. PBMC-CD19 CAR-T cells provide a non-durable benefit, which only delays disease onset. CONCLUSION: We identified characteristics of T cell stemness enriched in P-T CD19 CAR-T which are deficient in PBMC-derived products and translate into response durability in vivo. Our findings demonstrate that placental circulating T cells are a valuable cell source for allogeneic CAR-T products. Stemness advantages inherent to P-T cells translate to in vivo persistence advantages and long-term durable activity.

Bcl11b sustains multipotency and restricts effector programs of intestinal-resident memory CD8+ T cells.

The networks of transcription factors (TFs) that control intestinal-resident memory CD8+ T (TRM) cells, including multipotency and effector programs, are poorly understood. In this work, we investigated the role of the TF Bcl11b in TRM cells during infection with Listeria monocytogenes using mice with post-activation, conditional deletion of Bcl11b in CD8+ T cells. Conditional deletion of Bcl11b resulted in increased numbers of intestinal TRM cells and their precursors as well as decreased splenic effector and circulating memory cells and precursors. Loss of circulating memory cells was in part due to increased intestinal homing of Bcl11b-/- circulating precursors, with no major alterations in their programs. Bcl11b-/- TRM cells had altered transcriptional programs, with diminished expression of multipotent/multifunctional (MP/MF) program genes, including Tcf7, and up-regulation of the effector program genes, including Prdm1. Bcl11b also limits the expression of Ahr, another TF with a role in intestinal CD8+ TRM cell differentiation. Deregulation of TRM programs translated into a poor recall response despite TRM cell accumulation in the intestine. Reduced expression of MP/MF program genes in Bcl11b-/- TRM cells was linked to decreased chromatin accessibility and a reduction in activating histone marks at these loci. In contrast, the effector program genes displayed increased activating epigenetic status. These findings demonstrate that Bcl11b is a frontrunner in the tissue residency program of intestinal memory cells upstream of Tcf1 and Blimp1, promoting multipotency and restricting the effector program.

Design principles of microparticle size and immunomodulatory factor formulation dictate antigen-specific amelioration of multiple sclerosis in a mouse model.

Antigen-specific therapies allow for modulation of the immune system in a disease relevant context without systemic immune suppression. These therapies are especially valuable in autoimmune diseases such as multiple sclerosis (MS), where autoreactive T cells destroy myelin sheath. This work shows that an antigen-specific dual-sized microparticle (dMP) system can effectively halt and reverse disease progression in a mouse model of MS. Current MS treatments leave patients immunocompromised, but the dMP formulation spares the immune system as mice can successfully clear a Listeria Monocytogenes infection. Furthermore, we highlight design principles for particle based immunotherapies including the importance of delivering factors specific for immune cell recruitment (GM-CSF or SDF-1), differentiation (GM-CSF or FLT3L) and suppression (TGF-ß or VD3) in conjunction with disease relevant antigen, as the entire formulation is required for maximum efficacy. Lastly, the dMP scheme relies on formulating phagocytosable and non-phagocytosable MP sizes to direct payload to target either cell surface receptors or intracellular targets, as the reverse sized dMP formulation failed to reverse paralysis. We also challenge the design principles of the dMP system showing that the size of the MPs impact efficacy and that GM-CSF plays two distinct roles and that both of these must be replaced to match the primary effect of the dMP system. Overall, this work shows the versatile nature of the dMP system and expands the knowledge in particle science by emphasizing design tenets to guide the next generation of particle based immunotherapies.

Treatment with an antigen-specific dual microparticle system reverses advanced multiple sclerosis in mice.

Antigen-specific therapies hold promise for treating autoimmune diseases such as multiple sclerosis while avoiding the deleterious side effects of systemic immune suppression due to delivering the disease-specific antigen as part of the treatment. In this study, an antigen-specific dual-sized microparticle (dMP) treatment reversed hind limb paralysis when administered in mice with advanced experimental autoimmune encephalomyelitis (EAE). Treatment reduced central nervous system (CNS) immune cell infiltration, demyelination, and inflammatory cytokine levels. Mechanistic insights using single-cell RNA sequencing showed that treatment impacted the MHC II antigen presentation pathway in dendritic cells, macrophages, B cells, and microglia, not only in the draining lymph nodes but also strikingly in the spinal cord. CD74 and cathepsin S were among the common genes down-regulated in most antigen presenting cell (APC) clusters, with B cells also having numerous MHC II genes reduced. Efficacy of the treatment diminished when B cells were absent, suggesting their impact in this therapy, in concert with other immune populations. Activation and inflammation were reduced in both APCs and T cells. This promising antigen-specific therapeutic approach advantageously engaged essential components of both innate and adaptive autoimmune responses and capably reversed paralysis in advanced EAE without the use of a broad immunosuppressant.

CD177 modulates the function and homeostasis of tumor-infiltrating regulatory T cells.

Regulatory T (Treg) cells are one of the major immunosuppressive cell types in cancer and a potential target for immunotherapy, but targeting tumor-infiltrating (TI) Treg cells has been challenging. Here, using single-cell RNA sequencing of immune cells from renal clear cell carcinoma (ccRCC) patients, we identify two distinct transcriptional fates for TI Treg cells, Fate-1 and Fate-2. The Fate-1 signature is associated with a poorer prognosis in ccRCC and several other solid cancers. CD177, a cell surface protein normally expressed on neutrophil, is specifically expressed on Fate-1 TI Treg cells in several solid cancer types, but not on other TI or peripheral Treg cells. Mechanistically, blocking CD177 reduces the suppressive activity of Treg cells in vitro, while Treg-specific deletion of Cd177 leads to decreased tumor growth and reduced TI Treg frequency in mice. Our results thus uncover a functional CD177+ TI Treg population that may serve as a target for TI Treg-specific immunotherapy.

BCL11B is positioned upstream of PLZF and RORγt to control thymic development of mucosal-associated invariant T cells and MAIT17 program.

Mucosal-associated invariant T (MAIT) cells recognize microbial riboflavin metabolites presented by MR1 and play role in immune responses to microbial infections and tumors. We report here that absence of the transcription factor (TF) Bcl11b in mice alters predominantly MAIT17 cells in the thymus and further in the lung, both at steady state and following Salmonella infection. Transcriptomics and ChIP-seq analyses show direct control of TCR signaling program and position BCL11B upstream of essential TFs of MAIT17 program, including RORγt, ZBTB16 (PLZF), and MAF. BCL11B binding at key MAIT17 and at TCR signaling program genes in human MAIT cells occurred mostly in regions enriched for H3K27Ac. Unexpectedly, in human MAIT cells, BCL11B also bound at MAIT1 program genes, at putative active enhancers, although this program was not affected in mouse MAIT cells in the absence of Bcl11b. These studies endorse BCL11B as an essential TF for MAIT cells both in mice and humans.

The transcription factor Bcl11b promotes both canonical and adaptive NK cell differentiation.

Epigenetic landscapes can provide insight into regulation of gene expression and cellular diversity. Here, we examined the transcriptional and epigenetic profiles of seven human blood natural killer (NK) cell populations, including adaptive NK cells. The BCL11B gene, encoding a transcription factor (TF) essential for T cell development and function, was the most extensively regulated, with expression increasing throughout NK cell differentiation. Several Bcl11b-regulated genes associated with T cell signaling were specifically expressed in adaptive NK cell subsets. Regulatory networks revealed reciprocal regulation at distinct stages of NK cell differentiation, with Bcl11b repressing RUNX2 and ZBTB16 in canonical and adaptive NK cells, respectively. A critical role for Bcl11b in driving NK cell differentiation was corroborated in BCL11B-mutated patients and by ectopic Bcl11b expression. Moreover, Bcl11b was required for adaptive NK cell responses in a murine cytomegalovirus model, supporting expansion of these cells. Together, we define the TF regulatory circuitry of human NK cells and uncover a critical role for Bcl11b in promoting NK cell differentiation and function.

Bcl11b prevents fatal autoimmunity by promoting Treg cell program and constraining innate lineages in Treg cells.

Regulatory T (Treg) cells are essential for peripheral tolerance and rely on the transcription factor (TF) Foxp3 for their generation and function. Several other TFs are critical for the Treg cell program. We found that mice deficient in Bcl11b TF solely in Treg cells developed fatal autoimmunity, and Bcl11b-deficient Treg cells had severely altered function. Bcl11b KO Treg cells showed decreased functional marker levels in homeostatic conditions, inflammation, and tumors. Bcl11b controlled expression of essential Treg program genes at steady state and in inflammation. Bcl11b bound to genomic regulatory regions of Treg program genes in both human and mouse Treg cells, overlapping with Foxp3 binding; these genes showed altered chromatin accessibility in the absence of Bcl11b. Additionally, Bcl11b restrained myeloid and NK cell programs in Treg cells. Our study provides new mechanistic insights on the Treg cell program and identity control, with major implications for therapies in autoimmunity and cancer.

Hectd3 promotes pathogenic Th17 lineage through Stat3 activation and Malt1 signaling in neuroinflammation.

Polyubiquitination promotes proteasomal degradation, or signaling and localization, of targeted proteins. Here we show that the E3 ubiquitin ligase Hectd3 is necessary for pathogenic Th17 cell generation in experimental autoimmune encephalomyelitis (EAE), a mouse model for human multiple sclerosis. Hectd3-deficient mice have lower EAE severity, reduced Th17 program and inefficient Th17 cell differentiation. However, Stat3, but not RORγt, has decreased polyubiquitination, as well as diminished tyrosine-705 activating phosphorylation. Additionally, non-degradative polyubiquitination of Malt1, critical for NF-κB activation and Th17 cell function, is reduced. Mechanistically, Hectd3 promotes K27-linked and K29-linked polyubiquitin chains on Malt1, and K27-linked polyubiquitin chains on Stat3. Moreover, Stat3 K180 and Malt1 K648 are targeted by Hectd3 for non-degradative polyubiquitination to mediate robust generation of RORγt+IL-17Ahi effector CD4+ T cells. Thus, our studies delineate a mechanism connecting signaling related polyubiquitination of Malt1 and Stat3, leading to NF-kB activation and RORγt expression, to pathogenic Th17 cell function in EAE.

An antigen-specific semi-therapeutic treatment with local delivery of tolerogenic factors through a dual-sized microparticle system blocks experimental autoimmune encephalomyelitis.

Antigen-specific treatments are highly desirable for autoimmune diseases in contrast to treatments which induce systemic immunosuppression. A novel antigen-specific therapy has been developed which, when administered semi-therapeutically, is highly efficacious in the treatment of the mouse model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). The treatment uses dual-sized, polymeric microparticles (dMPs) loaded with specific antigen and tolerizing factors for intra- and extra-cellular delivery, designed to recruit and modulate dendritic cells toward a tolerogenic phenotype without systemic release. This approach demonstrated robust efficacy and provided complete protection against disease. Therapeutic efficacy required encapsulation of the factors in controlled-release microparticles and was antigen-specific. Disease blocking was associated with a reduction of infiltrating CD4+ T cells, inflammatory cytokine-producing pathogenic CD4+ T cells, and activated macrophages and microglia in the central nervous system. Furthermore, CD4+ T cells isolated from dMP-treated mice were anergic in response to disease-specific, antigen-loaded splenocytes. Additionally, the frequency of CD86hiMHCIIhi dendritic cells in draining lymph nodes of EAE mice treated with Ag-specific dMPs was reduced. Our findings highlight the efficacy of microparticle-based drug delivery platform to mediate antigen-specific tolerance, and suggest that such a multi-factor combinatorial approach can act to block autoimmunity.