Infection of mice by mouse cytomegalovirus (MCMV) triggers activation and expansion of Ly49H+ natural killer (NK) cells, which are virus specific and considered to be "adaptive" or "memory" NK cells. Here, we find that signaling lymphocytic activation molecule family receptors (SFRs), a group of hematopoietic cell-restricted receptors, are essential for the expansion of Ly49H+ NK cells after MCMV infection. This activity is largely mediated by CD48, an SFR broadly expressed on NK cells and displaying augmented expression after MCMV infection. It is also dependent on the CD48 counter-receptor, 2B4, expressed on host macrophages. The 2B4-CD48 axis promotes expansion of Ly49H+ NK cells by repressing their phagocytosis by virus-activated macrophages through inhibition of the pro-phagocytic integrin lymphocyte function-associated antigen-1 (LFA-1) on macrophages. These data identify key roles of macrophages and the 2B4-CD48 pathway in controlling the expansion of adaptive NK cells following MCMV infection. Stimulation of the 2B4-CD48 axis may be helpful in enhancing adaptive NK cell responses for therapeutic purposes.

MHC-independent αβTCRs (TCRs) recognize conformational epitopes on native self-proteins and arise in mice lacking both MHC and CD4/CD8 coreceptor proteins. Although naturally generated in the thymus, these TCRs resemble re-engineered therapeutic chimeric antigen receptor (CAR) T cells in their specificity for MHC-independent ligands. Here we identify naturally arising MHC-independent TCRs reactive to three native self-proteins (CD48, CD102, and CD155) involved in cell adhesion. We report that naturally arising MHC-independent TCRs require high affinity TCR-ligand engagements in the thymus to signal positive selection and that high affinity positive selection generates a peripheral TCR repertoire with limited diversity and increased self-reactivity. We conclude that the affinity of TCR-ligand engagements required to signal positive selection in the thymus inversely determines the diversity and self-tolerance of the mature TCR repertoire that is selected.

Natural killer (NK) cells eliminate virally infected and tumor cells. Among the receptors regulating NK cell function is 2B4 (CD244), a member of the signaling lymphocyte-activation molecule (SLAM) family that binds CD48. 2B4 is the only heterophilic receptor of the SLAM family, whose other members, e.g., NK-T-B-antigen (NTB-A), are self-ligands. We determined the structure of the complex between the N-terminal domains of mouse 2B4 and CD48, as well as the structures of unbound 2B4 and CD48. The complex displayed an association mode related to, yet distinct from, that of the NTB-A dimer. Binding was accompanied by the rigidification of flexible 2B4 regions containing most of the polymorphic residues across different species and receptor isoforms. We propose a model for 2B4-CD48 interactions that permits the intermixing of SLAM receptors with major histocompatibility complex-specific receptors in the NK cell immune synapse. This analysis revealed the basis for heterophilic recognition within the SLAM family.

Staining for CD27 and CD201 (endothelial protein C receptor) has been recently suggested as an alternative to stem cell antigen-1 (Sca1) to identify hematopoietic stem cells in inbred mouse strains with low or nil expression of SCA1. However, whether staining for CD27 and CD201 is compatible with low fms-like tyrosine kinase 3 (FLT3) expression and the "SLAM" code defined by CD48 and CD150 to identify mouse long-term reconstituting hematopoietic stem cells has not been established. We compared the C57BL/6 strain, which expresses a high level of SCA1 on hematopoietic stem cells to non-obese diabetic severe combined immune deficient NOD.CB17-prkdc scid/Sz (NOD-scid) mice and NOD.CB17-prkdc scid il2rg tm1Wj1/Sz (NSG) mice which both express low to negative levels of SCA1 on hematopoietic stem cells. We demonstrate that hematopoietic stem cells are enriched within the linage-negative C-KIT+ CD27+ CD201+ FLT3- CD48-CD150+ population in serial dilution long-term competitive transplantation assays. We also make the novel observation that CD48 expression is up-regulated in Lin- KIT+ progenitors from NOD-scid and NSG strains, which otherwise have very few cells expressing the CD48 ligand CD244. Finally, we report that unlike hematopoietic stem cells, SCA1 expression is similar on bone marrow endothelial and mesenchymal progenitor cells in C57BL/6, NOD-scid and NSG mice. In conclusion, we propose that the combination of Lineage, KIT, CD27, CD201, FLT3, CD48, and CD150 antigens can be used to identify long-term reconstituting hematopoietic stem cells from mouse strains expressing low levels of SCA1 on hematopoietic cells.

Hematopoietic stem and progenitor cells (HSPCs) are responsible for the production of blood and immune cells. Throughout life, HSPCs acquire oncogenic aberrations that can cause hematological cancers. Although molecular programs maintaining stem cell integrity have been identified, safety mechanisms eliminating malignant HSPCs from the stem cell pool remain poorly characterized. Here, we show that HSPCs constitutively present antigens via major histocompatibility complex class II. The presentation of immunogenic antigens, as occurring during malignant transformation, triggers bidirectional interactions between HSPCs and antigen-specific CD4+ T cells, causing stem cell proliferation, differentiation, and specific exhaustion of aberrant HSPCs. This immunosurveillance mechanism effectively eliminates transformed HSPCs from the hematopoietic system, thereby preventing leukemia onset. Together, our data reveal a bidirectional interaction between HSPCs and CD4+ T cells, demonstrating that HSPCs are not only passive receivers of immunological signals but also actively engage in adaptive immune responses to safeguard the integrity of the stem cell pool.

Inhibitory receptors mediate CD8 T-cell hyporesponsiveness against cancer and infectious diseases. PD-1 and CTLA-4 have been extensively studied, and blocking antibodies have already shown clinical benefit for cancer patients. Only little is known on extended co-expression of inhibitory receptors and their ligands. Here we analyzed the expression of eight inhibitory receptors by tumor-antigen specific CD8 T-cells. We found that the majority of effector T-cells simultaneously expressed four or more of the inhibitory receptors BTLA, TIM-3, LAG-3, KRLG-1, 2B4, CD160, PD-1 and CTLA-4. There were major differences depending on antigen-specificity, differentiation and anatomical localization of T-cells. On the other hand, naive T-cells were only single or double positive for BTLA and TIM-3. Extended co-expression is likely relevant for effector T-cells, as we found expression of multiple ligands in metastatic lesions of melanoma patients. Together, our data suggest that naive T-cells are primarily regulated by BTLA and TIM-3, whereas effector cells interact via larger numbers of inhibitory receptors. Blocking multiple inhibitory receptors simultaneously or sequentially may improve T-cell based therapies, but further studies are necessary to clarify the role of each receptor-ligand pair.

Natural killer (NK) cells are innate lymphocytes recognized for their important role against tumor cells. NK cells expressing chimeric antigen receptors (CARs) have enhanced effector function against various type of cancer and are attractive contenders for the next generation of cancer immunotherapies. However, a number of factors have hindered the application of NK cells for cellular therapy, including their poor in vitro growth kinetics and relatively low starting percentages within the mononuclear cell fraction of peripheral blood or cord blood (CB). To overcome these limitations, we genetically-engineered human leukocyte antigen (HLA)-A- and HLA-B- K562 cells to enforce the expression of CD48, 4-1BBL, and membrane-bound IL-21 (mbIL21), creating a universal antigen presenting cell (uAPC) capable of stimulating their cognate receptors on NK cells. We have shown that uAPC can drive the expansion of both non-transduced (NT) and CAR-transduced CB derived NK cells by >900-fold in 2 weeks of co-culture with excellent purity (>99.9%) and without indications of senescence/exhaustion. We confirmed that uAPC-expanded research- and clinical-grade NT and CAR-transduced NK cells have higher metabolic fitness and display enhanced effector function against tumor targets compared to the corresponding cell fractions cultured without uAPCs. This novel approach allowed the expansion of highly pure GMP-grade CAR NK cells at optimal cell numbers to be used for adoptive CAR NK cell-based cancer immunotherapy.

Many potential targets for CAR-T cells in solid tumors are expressed in some normal tissues, raising concern for off-tumor toxicity. Following lymphodepletion, CAR-T cells targeting the tumor-associated antigen ROR1 lysed tumors in mice but induced lethal bone marrow failure due to recognition of ROR1+ stromal cells. To improve selectivity, we engineered T cells with synthetic Notch (synNotch) receptors specific for EpCAM or B7-H3, which are expressed on ROR1+ tumor cells but not ROR1+ stromal cells. SynNotch receptors induced ROR1 CAR expression selectively within the tumor, resulting in tumor regression without toxicity when tumor cells were segregated from, but not when co-localized with, normal ROR1+ cells. This strategy, thus, permits safe targeting of tumors that are sufficiently separated from normal cells.

Supported lipid bilayers (SLBs) are one of the most common cell-membrane model systems to study cell-cell interactions. Nickel-chelating lipids are frequently used to functionalize the SLB with polyhistidine-tagged ligands. We show here that these lipids by themselves can induce calcium signaling in T cells, also when having protein ligands on the SLB. This is important to avoid "false" signaling events in cell studies with SLBs, but also to better understand the molecular mechanisms involved in T-cell signaling. Jurkat T cells transfected with the non-signaling molecule rat CD48 were found to bind to ligand-free SLBs containing ≥2 wt% nickel-chelating lipids upon which calcium signaling was induced. This signaling fraction steadily increased from 24 to 60% when increasing the amount of nickel-chelating lipids from 2 to 10 wt%. Both the signaling fraction and signaling time did not change significantly compared to ligand-free SLBs when adding the CD48-ligand rat CD2 to the SLB. Blocking the SLB with bovine serum albumin reduced the signaling fraction to 11%, while preserving CD2 binding and the exclusion of the phosphatase CD45 from the cell-SLB contacts. Thus, CD45 exclusion alone was not sufficient to result in calcium signaling. In addition, more cells signaled on ligand-free SLBs with copper-chelating lipids instead of nickel-chelating lipids and the signaling was found to be predominantly via T-cell receptor (TCR) triggering. Hence, it is possible that the nickel-chelating lipids act as ligands to the cell's TCRs, an interaction that needs to be blocked to avoid unwanted cell activation.

Diffuse large B-cell lymphoma (DLBCL) presents a great clinical challenge and has a poor prognosis, with immune-related genes playing a crucial role. We aimed to develop an immune-related prognostic signature for improving prognosis prediction in DLBCL. Samples from the GSE31312 dataset were randomly allocated to discovery and internal validation cohorts. Univariate Cox, random forest, LASSO regression and multivariate Cox analyses were utilized to develop a prognostic signature, which was verified in the internal validation cohort, entire validation cohort and external validation cohort (GSE10846). The tumor microenvironment was investigated using the CIBERSORT and ESTIMATE tools. Gene set enrichment analysis (GSEA) was further applied to analyze the entire GSE31312 cohort. We identified four immune-related genes (CD48, IL1RL, PSDM3, RXFP3) significantly associated with overall survival. Based on discovery and validation cohort analyses, this four-gene signature could classify patients into high- and low-risk groups, with significantly different prognoses. Activated memory CD4 T cells and activated dendritic cells were significantly decreased in the high-risk group, and these patients had lower immune scores. GSEA revealed enrichment of signaling pathways, such as T cell receptor, antigen receptor-mediated, antigen processing and presentation of peptide antigen via MHC class I, in the low-risk group. In conclusion, a robust signature based on four immune-related genes was successfully constructed for predicting prognosis in DLBCL patients.

Natural Killer (NK) cell responses to HIV-infected CD4 T cells (iCD4) depend on the integration of signals received through inhibitory (iNKR) and activating NK receptors (aNKR). iCD4 activate NK cells to inhibit HIV replication. HIV infection-dependent changes in the human leukocyte antigen (HLA) ligands for iNKR on iCD4 are well documented. By contrast, less is known regarding the HIV infection related changes in ligands for aNKR on iCD4. We examined the aNKR ligand profiles HIV p24⁺ HIV iCD4s that maintained cell surface CD4 (iCD4⁺), did not maintain CD4 (iCD4-) and uninfected CD4 (unCD4) T cells for expression of unique long (UL)-16 binding proteins-1 (ULBP-1), ULBP-2/5/6, ULBP-3, major histocompatibility complex (MHC) class 1-related (MIC)-A, MIC-B, CD48, CD80, CD86, CD112, CD155, Intercellular adhesion molecule (ICAM)-1, ICAM-2, HLA-E, HLA-F, HLA-A2, HLA-C, and the ligands to NKp30, NKp44, NKp46, and killer immunoglobulin-like receptor 3DS1 (KIR3DS1) by flow cytometry on CD4 T cells from 17 HIV-1 seronegative donors activated and infected with HIV. iCD4⁺ cells had higher expression of aNKR ligands than did unCD4. However, the expression of aNKR ligands on iCD4 where CD4 was downregulated (iCD4-) was similar to (ULBP-1, ULBP-2/5/6, ULBP-3, MIC-A, CD48, CD80, CD86 and CD155) or significantly lower than (MIC-B, CD112 and ICAM-2) what was observed on unCD4. Thus, HIV infection can be associated with increased expression of aNKR ligands or either baseline or lower than baseline levels of aNKR ligands, concomitantly with the HIV-mediated downregulation of cell surface CD4 on infected cells.

Adult stem cells are long-lived and quiescent with unique metabolic requirements. Macroautophagy/autophagy is a fundamental survival mechanism that allows cells to adapt to metabolic changes by degrading and recycling intracellular components. Here we address why autophagy depletion leads to a drastic loss of the stem cell compartment. Using inducible deletion of autophagy specifically in adult hematopoietic stem cells (HSCs) and in mice chimeric for autophagy-deficient and normal HSCs, we demonstrate that the stem cell loss is cell-intrinsic. Mechanistically, autophagy-deficient HSCs showed higher expression of several amino acid transporters (AAT) when compared to autophagy-competent cells, resulting in increased amino acid (AA) uptake. This was followed by sustained MTOR (mechanistic target of rapamycin) activation, with enlarged cell size, glucose uptake and translation, which is detrimental to the quiescent HSCs. MTOR inhibition by rapamycin treatment in vivo was able to rescue autophagy-deficient HSC loss and bone marrow failure and resulted in better reconstitution after transplantation. Our results suggest that targeting MTOR may improve aged stem cell function, promote reprogramming and stem cell transplantation.List of abbreviations: 5FU: fluoracil; AA: amino acids; AKT/PKB: thymoma viral proto-oncogene 1; ATF4: activating transcription factor 4; BafA: bafilomycin A1; BM: bone marrow; EIF2: eukaryotic initiation factor 2; EIF4EBP1/4EBP1: eukaryotic translation initiation factor 4E binding protein 1; KIT/CD117/c-Kit: KIT proto-oncogene receptor tyrosine kinase; HSCs: hematopoietic stem cells; HSPCs: hematopoietic stem and progenitor cells; Kyn: kynurenine; LSK: lineage- (Lin-), LY6A/Sca-1+, KIT/c-Kit/CD117+; LY6A/Sca-1: lymphocyte antigen 6 family member A; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; MTORC2: MTOR complex 2; OPP: O-propargyl-puromycin; PI3K: phosphoinositide 3-kinase; poly(I:C): polyinosinic:polycytidylic acid; RPS6/S6: ribosomal protein S6; tam: tamoxifen; TCA: tricarboxylic acid; TFEB: transcription factor EB; PTPRC/CD45: Protein Tyrosine Phosphatase Receptor Type C, CD45 antigen.

It has been proposed that CD2, which is highly expressed on T cells, serves to enhance T cell-antigen presenting cell (APC) adhesion and costimulate T cell activation. Here we analyzed the role of CD2 using CD2-deficient mice crossed with transgenic mice expressing a T cell receptor specific for lymphocytic choriomeningitis virus (LCMV)-derived peptide p33. We found that absence of CD2 on T cells shifted the p33-specific dose-response curve in vitro by a factor of 3-10. In comparison, stimulation of T cells in the absence of lymphocyte function-associated antigen (LFA)-1-intercellular adhesion molecule (ICAM)-1 interaction shifted the dose-response curve by a factor of 10, whereas absence of both CD2-CD48 and LFA-1-ICAM-1 interactions shifted the response by a factor of approximately 100. This indicates that CD2 and LFA-1 facilitate T cell activation additively. T cell activation at low antigen density was blocked at its very first steps, as T cell APC conjugate formation, TCR triggering, and Ca(2+) fluxes were affected by the absence of CD2. In vivo, LCMV-specific, CD2-deficient T cells proliferated normally upon infection with live virus but responded in a reduced fashion upon cross-priming. Thus, CD2 sets quantitative thresholds and fine-tunes T cell activation both in vitro and in vivo.

Pathogen-initiated chronic inflammation or autoimmune diseases accelerate proliferation and promote differentiation of hematopoietic stem cells (HSCs) but simultaneously reduce reconstitution capacity. Nevertheless, the effect of acute infection and inflammation on functional HSCs is still largely unknown. Here we found that acute infection elicited by heat-inactivated Escherichia coli (HIEC) expanded bone marrow lineage-negative (Lin)- stem-cell antigen 1 (Sca-1)+cKit+ (LSK) cell population, leading to reduced frequency of functional HSCs in LSK population. However, the total number of BM phenotypic HSCs (Flk2-CD48-CD150+ LSK cells) was not altered in HIEC-challenged mice. Additionally, the reconstitution capacity of the total BM between infected and uninfected mice was similar by both the competitive repopulation assay and measurement of functional HSCs by limiting dilution. Thus, occasionally occurring acute inflammation, which is critical for host defenses, is unlikely to affect HSC self-renewal and maintenance of long-term reconstitution capacity. During acute bacterial infection and inflammation, the hematopoietic system can replenish hematopoietic cells consumed in the innate inflammatory response by accelerating hematopoietic stem and progenitor cell proliferation, but preserving functional HSCs in the BM.

The relative contribution to cytotoxicity of each of the multiple NK cell activation receptors has been difficult to assess. Using Drosophila insect cells, which express ligands of human NK cell receptors, we show that target cell lysis by resting NK cells is controlled by different receptor signals for cytolytic granule polarization and degranulation. Intercellular adhesion molecule (ICAM)-1 on insect cells was sufficient to induce polarization of granules, but not degranulation, in resting NK cells. Conversely, engagement of the Fc receptor CD16 by rabbit IgG on insect cells induced degranulation without specific polarization. Lysis by resting NK cells occurred when polarization and degranulation were induced by the combined presence of ICAM-1 and IgG on insect cells. Engagement of receptor 2B4 by CD48 on insect cells induced weak polarization and no degranulation. However, coengagement of 2B4 and CD16 by their respective ligands resulted in granule polarization and cytotoxicity in the absence of leukocyte functional antigen-1-mediated adhesion to target cells. These data show that cytotoxicity by resting NK cells is controlled tightly by separate or cooperative signals from different receptors for granule polarization and degranulation.

Multiple sclerosis (MS) is an inflammatory CNS disease with a substantial genetic component, originally mapped to only the human leukocyte antigen (HLA) region. In the last 5 years, a total of seven genome-wide association studies and one meta-analysis successfully identified 57 non-HLA susceptibility loci. Here, we merged nominal statistical evidence of association and physical evidence of interaction to conduct a protein-interaction-network-based pathway analysis (PINBPA) on two large genetic MS studies comprising a total of 15,317 cases and 29,529 controls. The distribution of nominally significant loci at the gene level matched the patterns of extended linkage disequilibrium in regions of interest. We found that products of genome-wide significantly associated genes are more likely to interact physically and belong to the same or related pathways. We next searched for subnetworks (modules) of genes (and their encoded proteins) enriched with nominally associated loci within each study and identified those modules in common between the two studies. We demonstrate that these modules are more likely to contain genes with bona fide susceptibility variants and, in addition, identify several high-confidence candidates (including BCL10, CD48, REL, TRAF3, and TEC). PINBPA is a powerful approach to gaining further insights into the biology of associated genes and to prioritizing candidates for subsequent genetic studies of complex traits.

The gut microbiome shapes local and systemic immunity. The liver is presumed to be a protected sterile site. As such, a hepatic microbiome has not been examined. Here, we showed a liver microbiome in mice and humans that is distinct from that of the gut and is enriched in Proteobacteria. It undergoes dynamic alterations with age and is influenced by the environment and host physiology. Fecal microbial transfer experiments revealed that the liver microbiome is populated from the gut in a highly selective manner. Hepatic immunity is dependent on the microbiome, specifically the bacteroidetes species. Targeting bacteroidetes with oral antibiotics reduced hepatic immune cells by approximately 90%, prevented antigen-presenting cell (APC) maturation, and mitigated adaptive immunity. Mechanistically, our findings are consistent with presentation of bacteroidetes-derived glycosphingolipids to NKT cells promoting CCL5 signaling, which drives hepatic leukocyte expansion and activation, among other possible host-microbe interactions. Collectively, we reveal a microbial/glycosphingolipid/NKT/CCL5 axis that underlies hepatic immunity.

In the bone marrow, CXCL12 and IL-7 are essential for B cell differentiation, whereas hematopoietic stem cell (HSC) maintenance requires SCF and CXCL12. Peri-sinusoidal stromal (PSS) cells are the main source of IL-7, but their characterization as a pro-B cell niche remains limited. Here, we characterize pro-B cell supporting stromal cells and decipher the interaction network allowing pro-B cell retention. Preferential contacts are found between pro-B cells and PSS cells, which homogeneously express HSC and B cell niche genes. Furthermore, pro-B cells are frequently located in the vicinity of HSCs in the same niche. Using an interactome bioinformatics pipeline, we identify Nidogen-1 as essential for pro-B cell retention in the peri-sinusoidal niche as confirmed in Nidogen-1-/- mice. Finally, human pro-B cells and hematopoietic progenitors are observed close to similar IL-7+ stromal cells. Thus, a multispecific niche exists in mouse and human supporting both early progenitors and committed hematopoietic lineages.

B lymphocytes are exquisitely sensitive to fluctuations in nutrient signaling by the Rag GTPases, and 15% of follicular lymphomas (FLs) harbor activating mutations in RRAGC. Hence, a potential therapeutic approach against malignant B cells is to inhibit Rag GTPase signaling, but because such inhibitors are still to be developed, efficacy and safety remain unknown. We generated knockin mice expressing a hypomorphic variant of RagC (Q119L); RagCQ119L/+ mice are viable and show attenuated nutrient signaling. B lymphocyte activation is cell-intrinsically impaired in RagCQ119L/+ mice, which also show significant suppression of genetically induced lymphomagenesis and autoimmunity. Surprisingly, no overt systemic trade-offs or phenotypic alterations caused by partial suppression of nutrient signaling are seen in other organs, and RagCQ119L/+ mice show normal longevity and normal age-dependent health decline. These results support the efficacy and safety of moderate inhibition of nutrient signaling against pathological B cells.

2B4 (CD244) and NK-T-B-antigen (NTB-A, CD352) are activating receptors on human natural killer (NK) cells and belong to the family of signaling lymphocyte activation molecule (SLAM)-related receptors (SRR). Engagement of these receptors leads to phosphorylation of their cytoplasmic tails and recruitment of the adapter proteins SLAM-associated protein (SAP) and Ewing's sarcoma-activated transcript-2 (EAT-2). X-linked lymphoproliferative syndrome (XLP) is a severe immunodeficiency that results from mutations in the SAP gene. 2B4 and NTB-A-mediated cytotoxicity are abrogated in XLP NK cells. To elucidate the molecular basis for this defect we analyzed early signaling events in SAP knockdown cells. Similar to XLP NK cells, knockdown of SAP in primary human NK cells leads to a reduction of 2B4 and NTB-A-mediated cytotoxicity. We found that early signaling events such as raft recruitment and receptor phosphorylation are not affected by the absence of SAP, indicating the defect in the absence of SAP is downstream of these events. In addition, knockdown of EAT-2 does not impair 2B4 or NTB-A-mediated cytotoxicity. Surprisingly, EAT-2 recruitment to both receptors is abrogated in the absence of SAP, revealing a novel cooperativity between these adapters.