Quantitative reductions in T-cell receptor (TCR) signalling are associated with severe immunodeficiency, yet in certain cases can lead to autoimmunity. Mutation of the tyrosine kinase ZAP-70 can cause either of these outcomes, yet the limits of its signal transducing capacity are not well defined. To investigate these limits we have made use of mrtless: a chemically induced mutation of Zap70 associated with T-cell deficiency. Unlike cells devoid of ZAP-70, mrtless thymocytes showed partial induction of CD5 and CD69, and were sensitive to TCR stimulation with a dose-response shifted approximately 10-fold. However, essentially no T cells were able to compensate for the mrtless mutation and mature beyond the CD4⁺ CD8⁺ stage. This outcome contrasts with a ZAP-70 Src Homology 2 domain mutant strain, where high-affinity self-reactive TCR are positively selected rather than deleted. We discuss these data with respect to current models of TCR signalling in thymocyte selection.

IKAROS (encoded by IKZF1) is an important hematopoietic transcription factor critical for early B cell differentiation, with major defects known to lead to low B cell numbers and hypogammaglobulinemia. More perplexing is the link between IKZF1 variants and autoimmunity, including polymorphisms associated with susceptibility to SLE, and recently, rare variants driving monogenic autoimmunity. We identified a novel p.L188V mutation in IKZF1 in the index patient and her father and found this mutation to lead to loss of DNA binding. Peripheral B cells lacking a full complement of IKAROS function show upregulation of molecules accentuating B cell activation, while CD22, a key negative feedback circuit, is suppressed. The resulting hyperresponsiveness of peripheral B cells, in combination with elevated follicular helper T cell (Tfh) numbers, provides a putative mechanistic explanation for the association of IKZF1 variants with the emergence of autoimmune manifestations in this kindred.

Calcium signaling is essential for lymphocyte activation, with genetic disruptions of store-operated calcium (Ca2+) entry resulting in severe immunodeficiency. The inositol 1,4,5-trisphosphate receptor (IP3R), a homo- or heterotetramer of the IP3R1-3 isoforms, amplifies lymphocyte signaling by releasing Ca2+ from endoplasmic reticulum stores following antigen stimulation. Although knockout of all IP3R isoforms in mice causes immunodeficiency, the seeming redundancy of the isoforms is thought to explain the absence of variants in human immunodeficiency. In this study, we identified compound heterozygous variants of ITPR3 (a gene encoding IP3R subtype 3) in two unrelated Caucasian patients presenting with immunodeficiency. To determine whether ITPR3 variants act in a nonredundant manner and disrupt human immune responses, we characterized the Ca2+ signaling capacity, the lymphocyte response, and the clinical phenotype of these patients. We observed disrupted Ca2+ signaling in patient-derived fibroblasts and immune cells, with abnormal proliferation and activation responses following T-cell receptor stimulation. Reconstitution of IP3R3 in IP3R knockout cell lines led to the identification of variants as functional hypomorphs that showed reduced ability to discriminate between homeostatic and induced states, validating a genotype-phenotype link. These results demonstrate a functional link between defective endoplasmic reticulum Ca2+ channels and immunodeficiency and identify IP3Rs as diagnostic targets for patients with specific inborn errors of immunity. These results also extend the known cause of Ca2+-associated immunodeficiency from store-operated entry to impaired Ca2+ mobilization from the endoplasmic reticulum, revealing a broad sensitivity of lymphocytes to genetic defects in Ca2+ signaling.

FOXP3 deficiency results in severe multisystem autoimmunity in both mice and humans, driven by the absence of functional regulatory T cells. Patients typically present with early and severe autoimmune polyendocrinopathy, dermatitis, and severe inflammation of the gut, leading to villous atrophy and ultimately malabsorption, wasting, and failure to thrive. In the absence of successful treatment, FOXP3-deficient patients usually die within the first 2 years of life. Hematopoietic stem cell transplantation provides a curative option but first requires adequate control over the inflammatory condition. Due to the rarity of the condition, no clinical trials have been conducted, with widely unstandardized therapeutic approaches. We sought to compare the efficacy of lead therapeutic candidates rapamycin, anti-CD4 antibody, and CTLA4-Ig in controlling the physiological and immunological manifestations of Foxp3 deficiency in mice.

Foxp3+ regulatory T (Treg) cells of thymic (tTreg) and peripheral (pTreg) developmental origin are thought to synergistically act to ensure immune homeostasis, with self-reactive tTreg cells primarily constraining autoimmune responses. Here we exploited a Foxp3-dependent reporter with thymus-specific GFP/Cre activity to selectively ablate either tTreg (ΔtTreg) or pTreg (ΔpTreg) cell development, while sparing the respective sister populations. We found that, in contrast to the tTreg cell behavior in ΔpTreg mice, pTreg cells acquired a highly activated suppressor phenotype and replenished the Treg cell pool of ΔtTreg mice on a non-autoimmune C57BL/6 background. Despite the absence of tTreg cells, pTreg cells prevented early mortality and fatal autoimmunity commonly observed in Foxp3-deficient models of complete Treg cell deficiency, and largely maintained immune tolerance even as the ΔtTreg mice aged. However, only two generations of backcrossing to the autoimmune-prone non-obese diabetic (NOD) background were sufficient to cause severe disease lethality associated with different, partially overlapping patterns of organ-specific autoimmunity. This included a particularly severe form of autoimmune diabetes characterized by an early onset and abrogation of the sex bias usually observed in the NOD mouse model of human type 1 diabetes. Genetic association studies further allowed us to define a small set of autoimmune risk loci sufficient to promote β cell autoimmunity, including genes known to impinge on Treg cell biology. Overall, these studies show an unexpectedly high functional adaptability of pTreg cells, emphasizing their important role as mediators of bystander effects to ensure self-tolerance.

Mammalian genomes encode a plethora of long non-coding RNA (lncRNA). These transcripts are thought to regulate gene expression, influencing biological processes from development to pathology. Results from the few lncRNA that have been studied in the context of the immune system have highlighted potentially critical functions as network regulators. Here we explored the nature of the lncRNA transcriptome in regulatory T cells (Tregs), a subset of CD4+ T cells required to establish and maintain immunological self-tolerance. The identified Treg lncRNA transcriptome showed distinct differences from that of non-regulatory CD4+ T cells, with evidence of direct shaping of the lncRNA transcriptome by Foxp3, the master transcription factor driving the distinct mRNA profile of Tregs. Treg lncRNA changes were disproportionally reversed in the absence of Foxp3, with an enrichment for colocalisation with Foxp3 DNA binding sites, indicating a direct coordination of transcription by Foxp3 independent of the mRNA coordination function. We further identified a novel lncRNA Flatr, as a member of the core Treg lncRNA transcriptome. Flatr expression anticipates Foxp3 expression during in vitro Treg conversion, and Flatr-deficient mice show a mild delay in in vitro and peripheral Treg induction. These results implicate Flatr as part of the upstream cascade leading to Treg conversion, and may provide clues as to the nature of this process.

Hematopoietic stem cells (HSCs) self-renew in bone marrow niches formed by mesenchymal progenitors and endothelial cells expressing the chemokine CXCL12, but whether a separate niche instructs multipotent progenitor (MPP) differentiation remains unclear. We show that MPPs resided in HSC niches, where they encountered lineage-instructive differentiation signals. Conditional deletion of the chemokine receptor CXCR4 in MPPs reduced differentiation into common lymphoid progenitors (CLPs), which decreased lymphopoiesis. CXCR4 was required for CLP positioning near Interleukin-7+ (IL-7) cells and for optimal IL-7 receptor signaling. IL-7+ cells expressed CXCL12 and the cytokine SCF, were mesenchymal progenitors capable of differentiation into osteoblasts and adipocytes, and comprised a minor subset of sinusoidal endothelial cells. Conditional Il7 deletion in mesenchymal progenitors reduced B-lineage committed CLPs, while conditional Cxcl12 or Scf deletion from IL-7+ cells reduced HSC and MPP numbers. Thus, HSC maintenance and multilineage differentiation are distinct cell lineage decisions that are both controlled by HSC niches.

Interleukin 2 (IL-2) is a key homeostatic cytokine, with therapeutic applications in both immunogenic and tolerogenic immune modulation. Clinical use has been hampered by pleiotropic functionality and widespread receptor expression, with unexpected adverse events. Here, we developed a novel mouse strain to divert IL-2 production, allowing identification of contextual outcomes. Network analysis identified priority access for Tregs and a competitive fitness cost of IL-2 production among both Tregs and conventional CD4 T cells. CD8 T and NK cells, by contrast, exhibited a preference for autocrine IL-2 production. IL-2 sourced from dendritic cells amplified Tregs, whereas IL-2 produced by B cells induced two context-dependent circuits: dramatic expansion of CD8+ Tregs and ILC2 cells, the latter driving a downstream, IL-5-mediated, eosinophilic circuit. The source-specific effects demonstrate the contextual influence of IL-2 function and potentially explain adverse effects observed during clinical trials. Targeted IL-2 production therefore has the potential to amplify or quench particular circuits in the IL-2 network, based on clinical desirability.

Current immunotherapies provide limited benefits against T cell-depleted tumors, calling for therapeutic innovation. Using multi-omics integration of cancer patient data, we predict a type I interferon (IFN) responseHIGH state of dendritic cell (DC) vaccines, with efficacious clinical impact. However, preclinical DC vaccines recapitulating this state by combining immunogenic cancer cell death with induction of type I IFN responses fail to regress mouse tumors lacking T cell infiltrates. Here, in lymph nodes (LNs), instead of activating CD4+/CD8+ T cells, DCs stimulate immunosuppressive programmed death-ligand 1-positive (PD-L1+) LN-associated macrophages (LAMs). Moreover, DC vaccines also stimulate PD-L1+ tumor-associated macrophages (TAMs). This creates two anatomically distinct niches of PD-L1+ macrophages that suppress CD8+ T cells. Accordingly, a combination of PD-L1 blockade with DC vaccines achieves significant tumor regression by depleting PD-L1+ macrophages, suppressing myeloid inflammation, and de-inhibiting effector/stem-like memory T cells. Importantly, clinical DC vaccines also potentiate T cell-suppressive PD-L1+ TAMs in glioblastoma patients. We propose that a multimodal immunotherapy and vaccination regimen is mandatory to overcome T cell-depleted tumors.

The molecular cause of severe congenital neutropenia (SCN) is unknown in 30% to 50% of patients. SEC61A1 encodes the α-subunit of the Sec61 complex, which governs endoplasmic reticulum protein transport and passive calcium leakage. Recently, mutations in SEC61A1 were reported to be pathogenic in common variable immunodeficiency and glomerulocystic kidney disease.

NFIL3 is a key immunological transcription factor, with knockout mice studies identifying functional roles in multiple immune cell types. Despite the importance of NFIL3, little is known about its function in humans.

The lack of T cell infiltrates is a major obstacle to effective immunotherapy in cancer. Conversely, the formation of tumor-associated tertiary-lymphoid-like structures (TA-TLLSs), which are the local site of humoral and cellular immune responses against cancers, is associated with good prognosis, and they have recently been detected in immune checkpoint blockade (ICB)-responding patients. However, how these lymphoid aggregates develop remains poorly understood. By employing single-cell transcriptomics, endothelial fate mapping, and functional multiplex immune profiling, we demonstrate that antiangiogenic immune-modulating therapies evoke transdifferentiation of postcapillary venules into inflamed high-endothelial venules (HEVs) via lymphotoxin/lymphotoxin beta receptor (LT/LTβR) signaling. In turn, tumor HEVs boost intratumoral lymphocyte influx and foster permissive lymphocyte niches for PD1- and PD1+TCF1+ CD8 T cell progenitors that differentiate into GrzB+PD1+ CD8 T effector cells. Tumor-HEVs require continuous CD8 and NK cell-derived signals revealing that tumor HEV maintenance is actively sculpted by the adaptive immune system through a feed-forward loop.

The human CC chemokine receptor 8 (CCR8) is specifically expressed on tumor-infiltrating regulatory T cells (TITRs) and is a promising drug target for cancer immunotherapy. However, the role of CCR8 signaling in TITR biology and the effectiveness of CCR8 small molecule antagonists as TITR-targeting immunotherapy remain subjects of ongoing debate. In this work, we generated a novel cellular model of TITRs by culturing peripheral blood mononuclear cell-derived regulatory T cells in medium containing tumor cell-conditioned medium, CD3/CD28 activator, interleukin-2 and 1α,25-dihydroxyvitamin D3. This cellular model (named TITR mimics) highly and stably expressed a series of TITR signature molecules, including CCR8, FOXP3, CD30, CD39, CD134, CD137, TIGIT and Tim-3. Moreover, TITR mimics displayed robust in vitro immunosuppressive activity. To unravel the functional role of CCR8 in TITR mimics, a chemotaxis assay was performed showing strong and CCR8-specific migration toward CCL1, the natural chemokine agonist of CCR8. However, either stimulation (with CCL1) or blocking (with the small molecule antagonist NS-15) of CCR8 signaling did not affect the immunosuppressive activity, proliferation and survival of TITR mimics. Collectively, our work provides a method for the generation of TITR mimics in vitro, which can be used to study TITR biology and to evaluate drug candidates targeting TITRs. Furthermore, our findings suggest that CCR8 signaling primarily regulates migration of these cells.