CXCR4 signaling directs Igk recombination and the molecular mechanisms of late B lymphopoiesis.

In B lymphopoiesis, activation of the pre-B cell antigen receptor (pre-BCR) is associated with both cell cycle exit and Igk recombination. Yet how the pre-BCR mediates these functions remains unclear. Here, we demonstrate that the pre-BCR initiates a feed-forward amplification loop mediated by the transcription factor interferon regulatory factor 4 and the chemokine receptor C-X-C motif chemokine receptor 4 (CXCR4). CXCR4 ligation by C-X-C motif chemokine ligand 12 activates the mitogen-activated protein kinase extracellular-signal-regulated kinase, which then directs the development of small pre- and immature B cells, including orchestrating cell cycle exit, pre-BCR repression, Igk recombination and BCR expression. In contrast, pre-BCR expression and escape from interleukin-7 have only modest effects on B cell developmental transcriptional and epigenetic programs. These data show a direct and central role for CXCR4 in orchestrating late B cell lymphopoiesis. Furthermore, in the context of previous findings, our data provide a three-receptor system sufficient to recapitulate the essential features of B lymphopoiesis in vitro.

Chromothriptic cure of WHIM syndrome.

Chromothripsis is a catastrophic cellular event recently described in cancer in which chromosomes undergo massive deletion and rearrangement. Here, we report a case in which chromothripsis spontaneously cured a patient with WHIM syndrome, an autosomal dominant combined immunodeficiency disease caused by gain-of-function mutation of the chemokine receptor CXCR4. In this patient, deletion of the disease allele, CXCR4(R334X), as well as 163 other genes from one copy of chromosome 2 occurred in a hematopoietic stem cell (HSC) that repopulated the myeloid but not the lymphoid lineage. In competitive mouse bone marrow (BM) transplantation experiments, Cxcr4 haploinsufficiency was sufficient to confer a strong long-term engraftment advantage of donor BM over BM from either wild-type or WHIM syndrome model mice, suggesting a potential mechanism for the patient's cure. Our findings suggest that partial inactivation of CXCR4 may have general utility as a strategy to promote HSC engraftment in transplantation.

Gradients of the signaling lipid S1P in lymph nodes position natural killer cells and regulate their interferon-γ response.

The lymph node periphery is an important site for many immunological functions, from pathogen containment to the differentiation of helper T cells, yet the cues that position cells in this region are largely undefined. Here, through the use of a reporter for the signaling lipid S1P (sphingosine 1-phosphate), we found that cells sensed higher concentrations of S1P in the medullary cords than in the T cell zone and that the S1P transporter SPNS2 on lymphatic endothelial cells generated this gradient. Natural killer (NK) cells are located at the periphery of the lymph node, predominantly in the medulla, and we found that expression of SPNS2, expression of the S1P receptor S1PR5 on NK cells, and expression of the chemokine receptor CXCR4 were all required for NK cell localization during homeostasis and rapid production of interferon-γ by NK cells after challenge. Our findings elucidate the spatial cues for NK cell organization and reveal a previously unknown role for S1P in positioning cells within the medulla.

Defining Genetic Variation in Widely Used Congenic and Backcrossed Mouse Models Reveals Varied Regulation of Genes Important for Immune Responses.

Genetic variation influences how the genome is interpreted in individuals and in mouse strains used to model immune responses. We developed approaches to utilize next-generation sequencing datasets to identify sequence variation in genes and enhancer elements in congenic and backcross mouse models. We defined genetic variation in the widely used B6-CD45.2 and B6.SJL-CD45.1 congenic model, identifying substantial differences in SJL genetic content retained in B6.SJL-CD45.1 strains on the basis of the vendor source of the mice. Genes encoding PD-1, CD62L, Bcl-2, cathepsin E, and Cxcr4 were within SJL genetic content in at least one vendor source of B6.SJL-CD45.1 mice. SJL genetic content affected enhancer elements, gene regulation, protein expression, and amino acid content in CD4+ T helper 1 cells, and mice infected with influenza showed reduced expression of Cxcr4 on B6.SJL-CD45.1 T follicular helper cells. These findings provide information on experimental variables and aid in creating approaches that account for genetic variables.

Super-enhancer hypermutation alters oncogene expression in B cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) is the most common B cell non-Hodgkin lymphoma and remains incurable in around 40% of patients. Efforts to sequence the coding genome identified several genes and pathways that are altered in this disease, including potential therapeutic targets1-5. However, the non-coding genome of DLBCL remains largely unexplored. Here we show that active super-enhancers are highly and specifically hypermutated in 92% of samples from individuals with DLBCL, display signatures of activation-induced cytidine deaminase activity, and are linked to genes that encode B cell developmental regulators and oncogenes. As evidence of oncogenic relevance, we show that the hypermutated super-enhancers linked to the BCL6, BCL2 and CXCR4 proto-oncogenes prevent the binding and transcriptional downregulation of the corresponding target gene by transcriptional repressors, including BLIMP1 (targeting BCL6) and the steroid receptor NR3C1 (targeting BCL2 and CXCR4). Genetic correction of selected mutations restored repressor DNA binding, downregulated target gene expression and led to the counter-selection of cells containing corrected alleles, indicating an oncogenic dependency on the super-enhancer mutations. This pervasive super-enhancer mutational mechanism reveals a major set of genetic lesions deregulating gene expression, which expands the involvement of known oncogenes in DLBCL pathogenesis and identifies new deregulated gene targets of therapeutic relevance.

The development of brain pericytes requires expression of the transcription factor nkx3.1 in intermediate precursors.

Brain pericytes are one of the critical cell types that regulate endothelial barrier function and activity, thus ensuring adequate blood flow to the brain. The genetic pathways guiding undifferentiated cells into mature pericytes are not well understood. We show here that pericyte precursor populations from both neural crest and head mesoderm of zebrafish express the transcription factor nkx3.1 develop into brain pericytes. We identify the gene signature of these precursors and show that an nkx3.1-, foxf2a-, and cxcl12b-expressing pericyte precursor population is present around the basilar artery prior to artery formation and pericyte recruitment. The precursors later spread throughout the brain and differentiate to express canonical pericyte markers. Cxcl12b-Cxcr4 signaling is required for pericyte attachment and differentiation. Further, both nkx3.1 and cxcl12b are necessary and sufficient in regulating pericyte number as loss inhibits and gain increases pericyte number. Through genetic experiments, we have defined a precursor population for brain pericytes and identified genes critical for their differentiation.

Gain of Additional BIRC3 Protein Functions through 3'-UTR-Mediated Protein Complex Formation.

Alternative 3' untranslated regions (3' UTRs) are widespread, but their functional roles are largely unknown. We investigated the function of the long BIRC3 3' UTR, which is upregulated in leukemia. The 3' UTR does not regulate BIRC3 protein localization or abundance but is required for CXCR4-mediated B cell migration. We established an experimental pipeline to study the mechanism of regulation and used mass spectrometry to identify BIRC3 protein interactors. In addition to 3'-UTR-independent interactors involved in known BIRC3 functions, we detected interactors that bind only to BIRC3 protein encoded from the mRNA with the long 3' UTR. They regulate several functions, including CXCR4 trafficking. We further identified RNA-binding proteins differentially bound to the alternative 3' UTRs and found that cooperative binding of Staufen and HuR mediates 3'-UTR-dependent complex formation. We show that the long 3' UTR is required for the formation of specific protein complexes that enable additional functions of BIRC3 protein beyond its 3'-UTR-independent functions.

Increased Susceptibility of WHIM Mice to Papillomavirus-induced Disease is Dependent upon Immune Cell Dysfunction.

Warts, Hypogammaglobulinemia, Infections, and Myelokathexis (WHIM) syndrome is a rare primary immunodeficiency disease in humans caused by a gain of function in CXCR4, mostly due to inherited heterozygous mutations in CXCR4. One major clinical symptom of WHIM patients is their high susceptibility to human papillomavirus (HPV) induced disease, such as warts. Persistent high risk HPV infections cause 5% of all human cancers, including cervical, anogenital, head and neck and some skin cancers. WHIM mice bearing the same mutation identified in WHIM patients were created to study the underlying causes for the symptoms manifest in patients suffering from the WHIM syndrome. Using murine papillomavirus (MmuPV1) as an infection model in mice for HPV-induced disease, we demonstrate that WHIM mice are more susceptible to MmuPV1-induced warts (papillomas) compared to wild type mice. Namely, the incidence of papillomas is higher in WHIM mice compared to wild type mice when mice are exposed to low doses of MmuPV1. MmuPV1 infection facilitated both myeloid and lymphoid cell mobilization in the blood of wild type mice but not in WHIM mice. Higher incidence and larger size of papillomas in WHIM mice correlated with lower abundance of infiltrating T cells within the papillomas. Finally, we demonstrate that transplantation of bone marrow from wild type mice into WHIM mice normalized the incidence and size of papillomas, consistent with the WHIM mutation in hematopoietic cells contributing to higher susceptibility of WHIM mice to MmuPV1-induced disease. Our results provide evidence that MmuPV1 infection in WHIM mice is a powerful preclinical infectious model to investigate treatment options for alleviating papillomavirus infections in WHIM syndrome.

Multiomics analysis of IgM monoclonal gammopathies reveals epigenetic influence on oncogenesis via DNA methylation.

ABSTRACT: Currently, the role of DNA methylation in the immunoglobulin M (IgM) monoclonal gammopathy disease spectrum remains poorly understood. In the present study, a multiomics prospective analysis was conducted integrating DNA methylation, RNA sequencing (RNA-seq), and whole-exome sequencing data in 34 subjects (23 with Waldenström macroglobulinemia [WM], 6 with IgM monoclonal gammopathy of undetermined significance [MGUS], and 5 normal controls). Analysis was focused on defining differences between IgM gammopathies (WM/IgM-MGUS) compared with controls, and specifically between WM and IgM-MGUS. Between groups, genome-wide DNA methylation analysis demonstrated a significant number of differentially methylated regions that were annotated according to genomic region. Next, integration of RNA-seq data was performed to identify potentially epigenetically deregulated pathways. We found that pathways involved in cell cycle, metabolism, cytokine/immune signaling, cytoskeleton, tumor microenvironment, and intracellular signaling were differentially activated and potentially epigenetically regulated. Importantly, there was a positive enrichment of the CXCR4 signaling pathway along with several interleukin (interleukin 6 [IL-6], IL-8, and IL-15) signaling pathways in WM compared with IgM-MGUS. Further assessment of known tumor suppressor genes and oncogenes uncovered differential promoter methylation of several targets with concordant change in gene expression, including CCND1 and CD79B. Overall, this report defines how aberrant DNA methylation in IgM gammopathies may play a critical role in the epigenetic control of oncogenesis and key cellular functions.

How I use genomics and BTK inhibitors in the treatment of Waldenström macroglobulinemia.

ABSTRACT: Mutations in MYD88 (95%-97%) and CXCR4 (30%-40%) are common in Waldenström macroglobulinemia (WM). TP53 is altered in 20% to 30% of patients with WM, particularly those previously treated. Mutated MYD88 activates hematopoietic cell kinase that drives Bruton tyrosine kinase (BTK) prosurvival signaling. Both nonsense and frameshift CXCR4 mutations occur in WM. Nonsense variants show greater resistance to BTK inhibitors. Covalent BTK inhibitors (cBTKi) produce major responses in 70% to 80% of patients with WM. MYD88 and CXCR4 mutation status can affect time to major response, depth of response, and/or progression-free survival (PFS) in patients with WM treated with cBTKi. The cBTKi zanubrutinib shows greater response activity and/or improved PFS in patients with WM with wild-type MYD88, mutated CXCR4, or altered TP53. Risks for adverse events, including atrial fibrillation, bleeding diathesis, and neutropenia can differ based on which BTKi is used in WM. Intolerance is also common with cBTKi, and dose reduction or switchover to another cBTKi can be considered. For patients with acquired resistance to cBTKis, newer options include pirtobrutinib or venetoclax. Combinations of BTKis with chemoimmunotherapy, CXCR4, and BCL2 antagonists are discussed. Algorithms for positioning BTKis in treatment naïve or previously treated patients with WM, based on genomics, disease characteristics, and comorbidities, are presented.

Inducible CXCL12/CXCR4-dependent extramedullary hematopoietic niches in the adrenal gland.

ABSTRACT: Adult hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow (BM) hematopoietic niche, which regulates HSPC quiescence, self-renewal, and commitment in a demand-adapted manner. Although the complex BM niche is responsible for adult hematopoiesis, evidence exists for simpler, albeit functional and more accessible, extramedullary hematopoietic niches. Inspired by the anecdotal description of retroperitoneal hematopoietic masses occurring at higher frequency upon hormonal dysregulation within the adrenal gland, we hypothesized that the adult adrenal gland could be induced into a hematopoietic-supportive environment in a systematic manner, thus revealing mechanisms underlying de novo niche formation in the adult. Here, we show that upon splenectomy and hormonal stimulation, the adult adrenal gland of mice can be induced to recruit and host functional HSPCs, capable of serial transplantation, and that this phenomenon is associated with de novo formation of platelet-derived growth factor receptor α/leptin receptor (PDGFRα+/LEPR+/-)-expressing stromal nodules. We further show in CXCL12-green fluorescent protein reporter mice that adrenal glands contain a stromal population reminiscent of the CXCL12-abundant reticular cells, which compose the BM HSPC niche. Mechanistically, HSPC homing to hormonally induced adrenal glands was found dependent on the CXCR4-CXCL12 axis. Mirroring our findings in mice, we found reticular CXCL12+ cells coexpressing master niche regulator FOXC1 in primary samples from human adrenal myelolipomas, a benign tumor composed of adipose and hematopoietic tissue. Our findings reignite long-standing questions regarding hormonal regulation of hematopoiesis and provide a novel model to facilitate the study of adult-specific inducible hematopoietic niches, which may pave the way to therapeutic applications.

CCR6 Defines Memory B Cell Precursors in Mouse and Human Germinal Centers, Revealing Light-Zone Location and Predominant Low Antigen Affinity.

Memory B cells (MBCs) and plasma cells (PCs) constitute the two cellular outputs of germinal center (GC) responses that together facilitate long-term humoral immunity. Although expression of the transcription factor BLIMP-1 identifies cells undergoing PC differentiation, no such marker exists for cells committed to the MBC lineage. Here, we report that the chemokine receptor CCR6 uniquely marks MBC precursors in both mouse and human GCs. CCR6+ GC B cells were highly enriched within the GC light zone (LZ), were the most quiescent of all GC B cells, exhibited a cell-surface phenotype and gene expression signature indicative of an MBC transition, and possessed the augmented response characteristics of MBCs. MBC precursors within the GC LZ predominantly possessed a low affinity for antigen but also included cells from within the high-affinity pool. These data indicate a fundamental dichotomy between the processes that drive MBC and PC differentiation during GC responses.

Separating Actin-Dependent Chemokine Receptor Nanoclustering from Dimerization Indicates a Role for Clustering in CXCR4 Signaling and Function.

A current challenge in cell motility studies is to understand the molecular and physical mechanisms that govern chemokine receptor nanoscale organization at the cell membrane, and their influence on cell response. Using single-particle tracking and super-resolution microscopy, we found that the chemokine receptor CXCR4 forms basal nanoclusters in resting T cells, whose extent, dynamics, and signaling strength are modulated by the orchestrated action of the actin cytoskeleton, the co-receptor CD4, and its ligand CXCL12. We identified three CXCR4 structural residues that are crucial for nanoclustering and generated an oligomerization-defective mutant that dimerized but did not form nanoclusters in response to CXCL12, which severely impaired signaling. Overall, our data provide new insights to the field of chemokine biology by showing that receptor dimerization in the absence of nanoclustering is unable to fully support CXCL12-mediated responses, including signaling and cell function in vivo.

Genetic Deletion of Galectin-3 Inhibits Pancreatic Cancer Progression and Enhances the Efficacy of Immunotherapy.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) has a desmoplastic tumor stroma and immunosuppressive microenvironment. Galectin-3 (GAL3) is enriched in PDAC, highly expressed by cancer cells and myeloid cells. However, the functional roles of GAL3 in the PDAC microenvironment remain elusive. METHODS: We generated a novel transgenic mouse model (LSL-KrasG12D/+;Trp53loxP/loxP;Pdx1-Cre;Lgals3-/- [KPPC;Lgals3-/-]) that allows the genetic depletion of GAL3 from both cancer cells and myeloid cells in spontaneous PDAC formation. Single-cell RNA-sequencing analysis was used to identify the alterations in the tumor microenvironment upon GAL3 depletion. We investigated both the cancer cell-intrinsic function and immunosuppressive function of GAL3. We also evaluated the therapeutic efficacy of GAL3 inhibition in combination with immunotherapy. RESULTS: Genetic deletion of GAL3 significantly inhibited the spontaneous pancreatic tumor progression and prolonged the survival of KPPC;Lgals3-/- mice. Single-cell analysis revealed that genetic deletion of GAL3 altered the phenotypes of immune cells, cancer cells, and other cell populations. GAL3 deletion significantly enriched the antitumor myeloid cell subpopulation with high major histocompatibility complex class II expression. We also identified that GAL3 depletion resulted in CXCL12 upregulation, which could act as a potential compensating mechanism on GAL3 deficiency. Combined inhibition of the CXCL12-CXCR4 axis and GAL3 enhanced the efficacy of anti-PD-1 immunotherapy, leading to significantly inhibited PDAC progression. In addition, deletion of GAL3 also inhibited the basal/mesenchymal-like phenotype of pancreatic cancer cells. CONCLUSIONS: GAL3 promotes PDAC progression and immunosuppression via both cancer cell-intrinsic and immune-related mechanisms. Combined treatment targeting GAL3, CXCL12-CXCR4 axis, and PD-1 represents a novel therapeutic strategy for PDAC.

TGF-ß1-Induced SOX18 Elevation Promotes Hepatocellular Carcinoma Progression and Metastasis Through Transcriptionally Upregulating PD-L1 and CXCL12.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is characterized by an immune-suppressive microenvironment, which contributes to tumor progression, metastasis, and immunotherapy resistance. Identification of HCC-intrinsic factors regulating the immunosuppressive microenvironment is urgently needed. Here, we aimed to elucidate the role of SYR-Related High-Mobility Group Box 18 (SOX18) in inducing immunosuppression and to validate novel combination strategies for SOX18-mediated HCC progression and metastasis. METHODS: The role of SOX18 in HCC was investigated in orthotopic allografts and diethylinitrosamine/carbon tetrachloride-induced spontaneous models by using murine cell lines, adeno-associated virus 8, and hepatocyte-specific knockin and knockout mice. The immune cellular composition in the HCC microenvironment was evaluated by flow cytometry and immunofluorescence. RESULTS: SOX18 overexpression promoted the infiltration of tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) while diminishing cytotoxic T cells to facilitate HCC progression and metastasis in cell-derived allografts and chemically induced HCC models. Mechanistically, transforming growth factor-beta 1 (TGF-ß1) upregulated SOX18 expression by activating the Smad2/3 complex. SOX18 transactivated chemokine (C-X-C motif) ligand 12 (CXCL12) and programmed death ligand 1 (PD-L1) to induce the immunosuppressive microenvironment. CXCL12 knockdown significantly attenuated SOX18-induced TAMs and Tregs accumulation and HCC dissemination. Antagonism of chemokine receptor 4 (CXCR4), the cognate receptor of CXCL12, or selective knockout of CXCR4 in TAMs or Tregs likewise abolished SOX18-mediated effects. TGFßR1 inhibitor Vactosertib or CXCR4 inhibitor AMD3100 in combination with anti-PD-L1 dramatically inhibited SOX18-mediated HCC progression and metastasis. CONCLUSIONS: SOX18 promoted the accumulation of immunosuppressive TAMs and Tregs in the microenvironment by transactivating CXCL12 and PD-L1. CXCR4 inhibitor or TGFßR1 inhibitor in synergy with anti-PD-L1 represented a promising combination strategy to suppress HCC progression and metastasis.

Brain vascular damage-induced lymphatic ingrowth is directed by Cxcl12b/Cxcr4a.

After ischemic stroke, promotion of vascular regeneration without causing uncontrolled vessel growth appears to be the major challenge for pro-angiogenic therapies. The molecular mechanisms underlying how nascent blood vessels (BVs) are correctly guided into the post-ischemic infarction area remain unknown. Here, using a zebrafish cerebrovascular injury model, we show that chemokine signaling provides crucial guidance cues to determine the growing direction of ingrown lymphatic vessels (iLVs) and, in turn, that of nascent BVs. The chemokine receptor Cxcr4a is transcriptionally activated in the iLVs after injury, whereas its ligand Cxcl12b is expressed in the residual central BVs, the destinations of iLV ingrowth. Mutant and mosaic studies indicate that Cxcl12b/Cxcr4a-mediated chemotaxis is necessary and sufficient to determine the growing direction of iLVs and nascent BVs. This study provides a molecular basis for how the vessel directionality of cerebrovascular regeneration is properly determined, suggesting potential application of Cxcl12b/Cxcr4a in the development of post-ischemic pro-angiogenic therapies.

CRISPR/Cas9-mediated Cxcr4 disease allele inactivation for gene therapy in a mouse model of WHIM syndrome.

WHIM syndrome is an autosomal dominant immunodeficiency disorder caused by gain-of-function mutations in chemokine receptor CXCR4 that promote severe panleukopenia because of retention of mature leukocytes in the bone marrow (BM). We previously reported that Cxcr4-haploinsufficient (Cxcr4+/o) hematopoietic stem cells (HSCs) have a strong selective advantage for durable hematopoietic reconstitution over wild-type (Cxcr4+/+) and WHIM (Cxcr4+/w) HSCs and that a patient with WHIM was spontaneously cured by chromothriptic deletion of the disease allele in an HSC, suggesting that WHIM allele inactivation through gene editing may be a safe genetic cure strategy for the disease. We have developed a 2-step preclinical protocol of autologous hematopoietic stem and progenitor cell (HSPC) transplantation to achieve this goal. First, 1 copy of Cxcr4 in HSPCs was inactivated in vitro by CRISPR/Cas9 editing with a single guide RNA (sgRNA) that does not discriminate between Cxcr4+/w and Cxcr4+/+ alleles. Then, through in vivo natural selection, WHIM allele-inactivated cells were enriched over wild-type allele-inactivated cells. The WHIM allele-inactivated HSCs retained long-term pluripotency and selective hematopoietic reconstitution advantages. To our knowledge, this is the first example of gene therapy for an autosomal dominant gain-of-function disease using a disease allele inactivation strategy in place of the less efficient disease allele repair approach.

Severe CD8+ T Lymphopenia in WHIM Syndrome Caused by Selective Sequestration in Primary Immune Organs.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is an ultra-rare combined primary immunodeficiency disease caused by heterozygous gain-of-function mutations in the chemokine receptor CXCR4. WHIM patients typically present with recurrent acute infections associated with myelokathexis (severe neutropenia due to bone marrow retention of mature neutrophils). Severe lymphopenia is also common, but the only associated chronic opportunistic pathogen is human papillomavirus and mechanisms are not clearly defined. In this study, we show that WHIM mutations cause more severe CD8 than CD4 lymphopenia in WHIM patients and WHIM model mice. Mechanistic studies in mice revealed selective and WHIM allele dose-dependent accumulation of mature CD8 single-positive cells in thymus in a cell-intrinsic manner due to prolonged intrathymic residence, associated with increased CD8 single-positive thymocyte chemotactic responses in vitro toward the CXCR4 ligand CXCL12. In addition, mature WHIM CD8+ T cells preferentially home to and are retained in the bone marrow in mice in a cell-intrinsic manner. Administration of the specific CXCR4 antagonist AMD3100 (plerixafor) in mice rapidly and transiently corrected T cell lymphopenia and the CD4/CD8 ratio. After lymphocytic choriomeningitis virus infection, we found no difference in memory CD8+ T cell differentiation or viral load between wild-type and WHIM model mice. Thus, lymphopenia in WHIM syndrome may involve severe CXCR4-dependent CD8+ T cell deficiency resulting in part from sequestration in the primary lymphoid organs, thymus, and bone marrow.

Crosstalk between purinergic receptor P2Y11 and chemokine receptor CXCR7 is regulated by CXCR4 in human macrophages.

P2Y11 is a G protein-coupled ATP receptor that activates IL-1 receptor (IL-1R) in a cyclic AMP dependent manner. In human macrophages, P2Y11/IL-1R crosstalk with CCL20 as a prime target is controlled by phosphodiesterase 4 (PDE4), which mediates breakdown of cyclic AMP. Here, we used gene expression analysis to identify activation of CXCR4 and CXCR7 as a hallmark of P2Y11 signaling. We found that PDE4 inhibition with rolipram boosts P2Y11/IL-1R-induced upregulation of CXCR7 expression and CCL20 production in an epidermal growth factor receptor dependent manner. Using an astrocytoma cell line, naturally expressing CXCR7 but lacking CXCR4, P2Y11/IL-1R activation effectively induced and CXCR7 agonist TC14012 enhanced CCL20 production even in the absence of PDE4 inhibition. Moreover, CXCR7 depletion by RNA interference suppressed CCL20 production. In macrophages, the simultaneous activation of P2Y11 and CXCR7 by their respective agonists was sufficient to induce CCL20 production with no need of PDE4 inhibition, as CXCR7 activation increased its own and eliminated CXCR4 expression. Finally, analysis of multiple CCL chemokines in the macrophage secretome revealed that CXCR4 inactivation and CXCR7 activation selectively enhanced P2Y11/IL-1R-mediated secretion of CCL20. Altogether, our data establish CXCR7 as an integral component of the P2Y11/IL-1R-initiated signaling cascade and CXCR4-associated PDE4 as a regulatory checkpoint.

CD74 is a functional MIF receptor on activated CD4+ T cells.

Next to its classical role in MHC II-mediated antigen presentation, CD74 was identified as a high-affinity receptor for macrophage migration inhibitory factor (MIF), a pleiotropic cytokine and major determinant of various acute and chronic inflammatory conditions, cardiovascular diseases and cancer. Recent evidence suggests that CD74 is expressed in T cells, but the functional relevance of this observation is poorly understood. Here, we characterized the regulation of CD74 expression and that of the MIF chemokine receptors during activation of human CD4+ T cells and studied links to MIF-induced T-cell migration, function, and COVID-19 disease stage. MIF receptor profiling of resting primary human CD4+ T cells via flow cytometry revealed high surface expression of CXCR4, while CD74, CXCR2 and ACKR3/CXCR7 were not measurably expressed. However, CD4+ T cells constitutively expressed CD74 intracellularly, which upon T-cell activation was significantly upregulated, post-translationally modified by chondroitin sulfate and could be detected on the cell surface, as determined by flow cytometry, Western blot, immunohistochemistry, and re-analysis of available RNA-sequencing and proteomic data sets. Applying 3D-matrix-based live cell-imaging and receptor pathway-specific inhibitors, we determined a causal involvement of CD74 and CXCR4 in MIF-induced CD4+ T-cell migration. Mechanistically, proximity ligation assay visualized CD74/CXCR4 heterocomplexes on activated CD4+ T cells, which were significantly diminished after MIF treatment, pointing towards a MIF-mediated internalization process. Lastly, in a cohort of 30 COVID-19 patients, CD74 surface expression was found to be significantly upregulated on CD4+ and CD8+ T cells in patients with severe compared to patients with only mild disease course. Together, our study characterizes the MIF receptor network in the course of T-cell activation and reveals CD74 as a novel functional MIF receptor and MHC II-independent activation marker of primary human CD4+ T cells.