RBD-Based ELISA and Luminex Predict Anti-SARS-CoV-2 Surrogate-Neutralizing Activity in Two Longitudinal Cohorts of German and Spanish Health Care Workers.

The ability of antibodies to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important correlate of protection. For routine evaluation of protection, however, a simple and cost-efficient anti-SARS-CoV-2 serological assay predictive of serum neutralizing activity is needed. We analyzed clinical epidemiological data and blood samples from two cohorts of health care workers in Barcelona and Munich to compare several immunological readouts for evaluating antibody levels that could be surrogates of neutralizing activity. We measured IgG levels against SARS-CoV-2 spike protein (S), its S2 subunit, the S1 receptor binding domain (RBD), and the full length and C terminus of nucleocapsid (N) protein by Luminex, and against RBD by enzyme-linked immunosorbent assay (ELISA), and assessed those as predictors of plasma surrogate-neutralizing activity measured by a flow cytometry assay. In addition, we determined the clinical and demographic factors affecting plasma surrogate-neutralizing capacity. Both cohorts showed a high positive correlation between IgG levels to S antigen, especially to RBD, and the levels of plasma surrogate-neutralizing activity, suggesting RBD IgG as a good correlate of plasma neutralizing activity. Symptomatic infection, with symptoms such as loss of taste, dyspnea, rigors, fever and fatigue, was positively associated with anti-RBD IgG positivity by ELISA and Luminex, and with plasma surrogate-neutralizing activity. Our serological assays allow for the prediction of serum neutralization activity without the cost, hazards, time, and expertise needed for surrogate or conventional neutralization assays. Once a cutoff is established, these relatively simple high-throughput antibody assays will provide a fast and cost-effective method of assessing levels of protection from SARS-CoV-2 infection. IMPORTANCE Neutralizing antibody titers are the best correlate of protection against SARS-CoV-2. However, current tests to measure plasma or serum neutralizing activity do not allow high-throughput screening at the population level. Serological tests could be an alternative if they are proved to be good predictors of plasma neutralizing activity. In this study, we analyzed the SARS-CoV-2 serological profiles of two cohorts of health care workers by applying Luminex and ELISA in-house serological assays. Correlations of both serological tests were assessed between them and with a flow cytometry assay to determine plasma surrogate-neutralizing activity. Both assays showed a high positive correlation between IgG levels to S antigens, especially RBD, and the levels of plasma surrogate-neutralizing activity. This result suggests IgG to RBD as a good correlate of plasma surrogate-neutralizing activity and indicates that serology of IgG to RBD could be used to assess levels of protection from SARS-CoV-2 infection.

Autophagy-mediated NCOR1 degradation is required for brown fat maturation and thermogenesis.

Brown adipose tissue (BAT) thermogenesis affects energy balance, and thereby it has the potential to induce weight loss and to prevent obesity. Here, we document a macroautophagic/autophagic-dependent mechanism of peroxisome proliferator-activated receptor gamma (PPARG) activity regulation that induces brown adipose differentiation and thermogenesis and that is mediated by TP53INP2. Disruption of TP53INP2-dependent autophagy reduced brown adipogenesis in cultured cells. In vivo specific-tp53inp2 ablation in brown precursor cells or in adult mice decreased the expression of thermogenic and mature adipocyte genes in BAT. As a result, TP53INP2-deficient mice had reduced UCP1 content in BAT and impaired maximal thermogenic capacity, leading to lipid accumulation and to positive energy balance. Mechanistically, TP53INP2 stimulates PPARG activity and adipogenesis in brown adipose cells by promoting the autophagic degradation of NCOR1, a PPARG co-repressor. Moreover, the modulation of TP53INP2 expression in BAT and in human brown adipocytes suggests that this protein increases PPARG activity during metabolic activation of brown fat. In all, we have identified a novel molecular explanation for the contribution of autophagy to BAT energy metabolism that could facilitate the design of therapeutic strategies against obesity and its metabolic complications.

Experimental and genetic evidence for the impact of CD5 and CD6 expression and variation in inflammatory bowel disease.

Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) resulting from the interaction of multiple environmental, genetic and immunological factors. CD5 and CD6 are paralogs encoding lymphocyte co-receptors involved in fine-tuning intracellular signals delivered upon antigen-specific recognition, microbial pattern recognition and cell adhesion. While CD5 and CD6 expression and variation is known to influence some immune-mediated inflammatory disorders, their role in IBD remains unclear. To this end, Cd5- and Cd6-deficient mice were subjected to dextran sulfate sodium (DSS)-induced colitis, the most widely used experimental animal model of IBD. The two mouse lines showed opposite results regarding body weight loss and disease activity index (DAI) changes following DSS-induced colitis, thus supporting Cd5 and Cd6 expression involvement in the pathophysiology of this experimental IBD model. Furthermore, DNA samples from IBD patients of the ENEIDA registry were used to test association of CD5 (rs2241002 and rs2229177) and CD6 (rs17824933, rs11230563, and rs12360861) single nucleotide polymorphisms with susceptibility and clinical parameters of CD (n=1352) and UC (n=1013). Generalized linear regression analyses showed association of CD5 variation with CD ileal location (rs2241002CC) and requirement of biological therapies (rs2241002C-rs2229177T haplotype), and with poor UC prognosis (rs2241002T-rs2229177T haplotype). Regarding CD6, association was observed with CD ileal location (rs17824933G) and poor prognosis (rs12360861G), and with left-sided or extensive UC, and absence of ankylosing spondylitis in IBD (rs17824933G). The present experimental and genetic evidence support a role for CD5 and CD6 expression and variation in IBD's clinical manifestations and therapeutic requirements, providing insight into its pathophysiology and broadening the relevance of both immunomodulatory receptors in immune-mediated disorders.

CD6 deficiency impairs early immune response to bacterial sepsis.

CD6 is a lymphocyte-specific scavenger receptor expressed on adaptive (T) and innate (B1a, NK) immune cells, which is involved in both fine-tuning of lymphocyte activation/differentiation and recognition of bacterial-associated molecular patterns (i.e., lipopolysaccharide). However, evidence on CD6's role in the physiological response to bacterial infection was missing. Our results show that induction of monobacterial and polymicrobial sepsis in Cd6 -/- mice results in lower survival rates and increased bacterial loads and pro-inflammatory cytokine levels. Steady state analyses of Cd6 -/- mice show decreased levels of natural polyreactive antibodies, concomitant with decreased cell counts of spleen B1a and marginal zone B cells. Adoptive transfer of wild-type B cells and mouse serum, as well as a polyreactive monoclonal antibody improve Cd6 -/- mouse survival rates post-sepsis. These findings support a nonredundant role for CD6 in the early response against bacterial infection, through homeostatic expansion and functionality of innate-related immune cells.

Discovery of the first PD-1 ligand encoded by a pathogen.

Large double-stranded DNA viruses deploy multiple strategies to subvert host immune defenses. Some of these tactics are mediated by viral gene products acquired by horizontal gene transfer from the corresponding hosts and shaped throughout evolution. The programmed death-1 (PD-1) receptor and its ligands, PD-L1 and PD-L2, play a pivotal role attenuating T-cell responses and regulating immune tolerance. In this study, we report the first functional PD-L1 homolog gene (De2) found in a pathogen. De2, captured by a γ-herpesvirus from its host during co-evolution around 50 million years ago, encodes a cell-surface glycoprotein that interacts with high affinity and stability with host PD-1. We also find that mutations evolved by the viral protein result in a significant loss of its ability to interact in cis with CD80, an interaction that for PD-L1:CD80 has been reported to block PD-1 inhibitory pathways. Furthermore, we demonstrate that the viral protein strongly inhibits T-cell signaling. Our observations suggest that PD-L1 homologs may enable viruses to evade T cell responses, favor their replication, and prevent excessive tissue damage. Altogether, our findings reveal a novel viral immunosuppressive strategy and highlight the importance of the modulation of the PD-1/PD-L1 axis during viral infections.

CD229 (Ly9) a Novel Biomarker for B-Cell Malignancies and Multiple Myeloma.

CD229 (Ly9) homophilic receptor, which belongs to the SLAM family of cell-surface molecules, is predominantly expressed on B and T cells. It acts as a signaling molecule, regulating lymphocyte homoeostasis and activation. Studies of CD229 function indicate that this receptor functions as a regulator of the development of marginal-zone B cells and other innate-like T and B lymphocytes. The expression on leukemias and lymphomas remains poorly understood due to the lack of CD229 monoclonal antibodies (mAb) for immunohistochemistry application (IHC). In this study, we used a new mAb against the cytoplasmic region of CD229 to study the expression of CD229 on normal tissues and B-cell malignancies, including multiple myeloma (MM), using tissue microarrays. We showed CD229 to be restricted to hematopoietic cells. It was strongly expressed in all cases of MM and in most marginal-zone lymphomas (MZL). Moderate CD229 expression was also found in chronic lymphocyte leukemia (CLL), follicular (FL), classic mantle-cell (MCL) and diffuse large B-cell lymphoma. Given the high expression on myeloma cells, we also analyzed for the presence of soluble CD229 in the sera of these patients. Serum levels of soluble CD229 (sCD229) at the time of diagnosis in MM patients could be useful as a prognostic biomarker. In conclusion, our results indicate that CD229 represents not only a useful biomarker but also an attractive therapeutic target.

Decreased and Heterogeneous Neutralizing Antibody Responses Against RBD of SARS-CoV-2 Variants After mRNA Vaccination.

The rapid spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerging variants raises concerns about their capacity to evade immune protection provided by natural infection or vaccination. The receptor-binding domain (RBD) of the viral spike protein is the major target of neutralizing antibodies, and viral variants accumulate mutations in this region. In this study, we determined the antibody neutralization capacity against the RBD of SARS-CoV-2 variants Alpha (B.1.1.7), Gamma (P.1), Epsilon (B.1.427), Kappa (B.1.617.1), and Delta (B.1.617.2) in a cohort of healthcare workers naturally infected or receiving COVID-19 mRNA vaccines from Moderna or Pfizer-BioNTech. We show that the five RBD variants displayed an augmented binding to ACE2 compared to the original Wuhan strain. The most significant increase was observed in variants Epsilon and Delta, containing mutation L452R. Using a flow cytometry cell-based assay, we found that SARS-CoV-2-infected subjects presented low levels of RBD-specific neutralizing antibodies against all variants analyzed, except Alpha. However, the neutralizing activity incremented considerably after a subsequent mRNA-vaccine dose, to levels significantly higher than those in naïve individuals receiving two vaccine doses. Importantly, we observed partially impaired neutralizing responses against most variants in fully vaccinated individuals. Variants Gamma and Kappa encompassing RBD E484K/Q mutations presented the highest neutralizing resistance. Furthermore, a wide heterogeneity in the magnitude of RBD-specific neutralizing responses against all tested SARS-CoV-2 variants following both mRNA vaccines was detected. Altogether, our findings provide important knowledge regarding SARS-CoV-2 vaccine-induced immunity, and should be very useful to guide future vaccination regimens and personalized vaccine approaches.

SIRPα - CD47 axis regulates dendritic cell-T cell interactions and TCR activation during T cell priming in spleen.

The SIRPα-CD47 axis plays an important role in T cell recruitment to sites of immune reaction and inflammation but its role in T cell antigen priming is incompletely understood. Employing OTII TCR transgenic mice bred to Cd47-/- (Cd47KO) or SKI mice, a knock-in transgenic animal expressing non-signaling cytoplasmic-truncated SIRPα, we investigated how the SIRPα-CD47 axis contributes to antigen priming. Here we show that adoptive transfer of Cd47KO or SKI Ova-specific CD4+ T cells (OTII) into Cd47KO and SKI recipients, followed by Ova immunization, elicited reduced T cell division and proliferation indices, increased apoptosis, and reduced expansion compared to transfer into WT mice. We confirmed prior reports that splenic T cell zone, CD4+ conventional dendritic cells (cDCs) and CD4+ T cell numbers were reduced in Cd47KO and SKI mice. We report that in vitro derived DCs from Cd47KO and SKI mice exhibited impaired migration in vivo and exhibited reduced CD11c+ DC proximity to OTII T cells in T cell zones after Ag immunization, which correlates with reduced TCR activation in transferred OTII T cells. These findings suggest that reduced numbers of CD4+ cDCs and their impaired migration contributes to reduced T cell-DC proximity in splenic T cell zone and reduced T cell TCR activation, cell division and proliferation, and indirectly increased T cell apoptosis.

Standardization of Workflow and Flow Cytometry Panels for Quantitative Expression Profiling of Surface Antigens on Blood Leukocyte Subsets: An HCDM CDMaps Initiative.

Background: The Human Cell Differentiation Molecules (HCDM) organizes Human Leukocyte Differentiation Antigen (HLDA) workshops to test and name clusters of antibodies that react with a specific antigen. These cluster of differentiation (CD) markers have provided the scientific community with validated antibody clones, consistent naming of targets and reproducible identification of leukocyte subsets. Still, quantitative CD marker expression profiles and benchmarking of reagents at the single-cell level are currently lacking. Objective: To develop a flow cytometric procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets that is standardized across multiple research laboratories. Methods: A high content framework to evaluate the titration and reactivity of Phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) was created. Two flow cytometry panels were designed: an innate cell tube for granulocytes, dendritic cells, monocytes, NK cells and innate lymphoid cells (12-color) and an adaptive lymphocyte tube for naive and memory B and T cells, including TCRγδ+, regulatory-T and follicular helper T cells (11-color). The potential of these 2 panels was demonstrated via expression profiling of selected CD markers detected by PE-conjugated antibodies and evaluated using 561 nm excitation. Results: Using automated data annotation and dried backbone reagents, we reached a robust workflow amenable to processing hundreds of measurements in each experiment in a 96-well plate format. The immunophenotyping panels enabled discrimination of 27 leukocyte subsets and quantitative detection of the expression of PE-conjugated CD markers of interest that could quantify protein expression above 400 units of antibody binding capacity. Expression profiling of 4 selected CD markers (CD11b, CD31, CD38, CD40) showed high reproducibility across centers, as well as the capacity to benchmark unique clones directed toward the same CD3 antigen. Conclusion: We optimized a procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets. The workflow, bioinformatics pipeline and optimized flow panels enable the following: 1) mapping the expression patterns of HLDA-approved mAb clones to CD markers; 2) benchmarking new antibody clones to established CD markers; 3) defining new clusters of differentiation in future HLDA workshops.

Determinants of early antibody responses to COVID-19 mRNA vaccines in a cohort of exposed and naïve healthcare workers.

BACKGROUND: Two doses of mRNA vaccination have shown >94% efficacy at preventing COVID-19 mostly in naïve adults, but it is not clear if the second dose is needed to maximize effectiveness in those previously exposed to SARS-CoV-2 and what other factors affect responsiveness. METHODS: We measured IgA, IgG and IgM levels against SARS-CoV-2 spike (S) and nucleocapsid (N) antigens from the wild-type and S from the Alpha, Beta and Gamma variants of concern, after BNT162b2 (Pfizer/BioNTech) or mRNA-1273 (Moderna) vaccination in a cohort of health care workers (N=578). Neutralizing capacity and antibody avidity were evaluated. Data were analyzed in relation to COVID-19 history, comorbidities, vaccine doses, brand and adverse events. FINDINGS: Vaccination induced robust IgA and IgG levels against all S antigens. Neutralization capacity and S IgA and IgG levels were higher in mRNA-1273 vaccinees, previously SARS-CoV-2 exposed, particularly if symptomatic, and in those experiencing systemic adverse effects (p<0·05). A second dose in pre-exposed did not increase antibody levels. Smoking and comorbidities were associated with 43% (95% CI, 19-59) and 45% (95% CI, 63-18) lower neutralization, respectively, and 35% (95% CI, 3-57%) and 55% (95% CI, 33-70%) lower antibody levels, respectively. Among fully vaccinated, 6·3% breakthroughs were detected up to 189 days post-vaccination. Among pre-exposed non-vaccinated, 90% were IgG seropositive more than 300 days post-infection. INTERPRETATION: Our data support administering a single-dose in pre-exposed healthy individuals as primary vaccination. However, heterogeneity of responses suggests that personalized recommendations may be necessary depending on COVID-19 history and life-style. Higher mRNA-1273 immunogenicity would be beneficial for those expected to respond worse to vaccination and in face of variants that escape immunity such as Omicron. Persistence of antibody levels in pre-exposed unvaccinated indicates maintenance of immunity up to one year. FUNDING: This work was supported by Institut de Salut Global de Barcelona (ISGlobal) internal funds, in-kind contributions from Hospital Clínic de Barcelona, the Fundació Privada Daniel Bravo Andreu, and European Institute of Innovation and Technology (EIT) Health (grant number 20877), supported by the European Institute of Innovation and Technology, a body of the European Union receiving support from the H2020 Research and Innovation Programme. We acknowledge support from the Spanish Ministry of Science and Innovation and State Research Agency through the "Centro de Excelencia Severo Ochoa 2019-2023" Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. L. I. work was supported by PID2019-110810RB-I00 grant from the Spanish Ministry of Science & Innovation. Development of SARS-CoV-2 reagents was partially supported by the National Institute of Allergy and Infectious Diseases Centers of Excellence for Influenza Research and Surveillance (contract number HHSN272201400008C). The funders had no role in study design, data collection and analysis, the decision to publish, or the preparation of the manuscript.

Characterization of Novel P-Selectin Targeted Complement Inhibitors in Murine Models of Hindlimb Injury and Transplantation.

The complement system has long been recognized as a potential druggable target for a variety of inflammatory conditions. Very few complement inhibitors have been approved for clinical use, but a great number are in clinical development, nearly all of which systemically inhibit complement. There are benefits of targeting complement inhibition to sites of activation/disease in terms of efficacy and safety, and here we describe P-selectin targeted complement inhibitors, with and without a dual function of directly blocking P-selectin-mediated cell-adhesion. The constructs are characterized in vitro and in murine models of hindlimb ischemia/reperfusion injury and hindlimb transplantation. Both constructs specifically targeted to reperfused hindlimb and provided protection in the hindlimb ischemia/reperfusion injury model. The P-selectin blocking construct was the more efficacious, which correlated with less myeloid cell infiltration, but with similarly reduced levels of complement deposition. The blocking construct also improved tissue perfusion and, unlike the nonblocking construct, inhibited coagulation, raising the possibility of differential application of each construct, such as in thrombotic vs. hemorrhagic conditions. Similar outcomes were obtained with the blocking construct following vascularized composite graft transplantation, and treatment also significantly increased graft survival. This is outcome may be particularly pertinent in the context of vascularized composite allograft transplantation, since reduced ischemia reperfusion injury is linked to a less rigorous alloimmune response that may translate to the requirement of a less aggressive immunosuppressive regime for this normally nonlife-threatening procedure. In summary, we describe a new generation of targeted complement inhibitor with multi-functionality that includes targeting to vascular injury, P-selectin blockade, complement inhibition and anti-thrombotic activity. The constructs described also bound to both mouse and human P-selectin which may facilitate potential translation.

Seven-month kinetics of SARS-CoV-2 antibodies and role of pre-existing antibodies to human coronaviruses.

Unraveling the long-term kinetics of antibodies to SARS-CoV-2 and the individual characteristics influencing it, including the impact of pre-existing antibodies to human coronaviruses causing common cold (HCoVs), is essential to understand protective immunity to COVID-19 and devise effective surveillance strategies. IgM, IgA and IgG levels against six SARS-CoV-2 antigens and the nucleocapsid antigen of the four HCoV (229E, NL63, OC43 and HKU1) were quantified by Luminex, and antibody neutralization capacity was assessed by flow cytometry, in a cohort of health care workers followed up to 7 months (N = 578). Seroprevalence increases over time from 13.5% (month 0) and 15.6% (month 1) to 16.4% (month 6). Levels of antibodies, including those with neutralizing capacity, are stable over time, except IgG to nucleocapsid antigen and IgM levels that wane. After the peak response, anti-spike antibody levels increase from ~150 days post-symptom onset in all individuals (73% for IgG), in the absence of any evidence of re-exposure. IgG and IgA to HCoV are significantly higher in asymptomatic than symptomatic seropositive individuals. Thus, pre-existing cross-reactive HCoVs antibodies could have a protective effect against SARS-CoV-2 infection and COVID-19 disease.

SOX11, CD70, and Treg cells configure the tumor-immune microenvironment of aggressive mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a mature B-cell neoplasm with a heterogeneous clinical and biological behavior. SOX11 oncogenic expression contributes to the aggressiveness of these tumors by different mechanisms, including tumor and stromal cell interactions. However, the precise composition of the immune cell microenvironment of MCL, its possible relationship to SOX11 expression, and how it may contribute to tumor behavior is not well known. Here, we performed an integrative transcriptome analysis of 730 immune-related genes combined with the immune cell phenotype analysis by immunohistochemistry in SOX11+ and SOX11- primary nodal MCL cases and non-neoplastic reactive lymph nodes. SOX11+ MCL had a significant lower T-cell intratumoral infiltration compared with negative cases. A reduced expression of MHCI/II-like and T-cell costimulation and signaling activation related transcripts was significantly associated with poor clinical outcome. Moreover, we identified CD70 as a SOX11 direct target gene, whose overexpression was induced in SOX11+, but not SOX11- tumor cells by CD40L in vitro. CD70 was overexpressed in primary SOX11+ MCL and it was associated with an immune unbalance of the tumor microenvironment characterized by increased number of effector regulatory t (Treg) cell infiltration, higher proliferation, and aggressive clinical course. CD27 was expressed with moderate to strong intensity in 76% of cases. Overall, our results suggest that SOX11 expression in MCL is associated with an immunosuppressive microenvironment characterized by CD70 overexpression in tumor cells, increased Treg cell infiltration and downmodulation of antigen processing, and presentation and T-cell activation that could promote MCL progression and represent a potential target for tailored therapies.

Autoimmune B Cell Repertoire in a Mouse Model of Sjögren's Syndrome.

In genetically prone individuals, chronic immune activation may lead to expansion of autoreactive lymphocyte clones that can induce organ damage developing autoimmune disorders. Sjögren's Syndrome (SjS) is a systemic chronic autoimmune disease that primarily affects exocrine glands. Despite the accumulated evidences of profound B-cell alterations of humoral immunity, the repertoire and development of B-cell autoreactivity in SjS remains to be determined. We hypothesize that SjS mice will have an increased frequency of self-reactive B cells with a progressive evolution to antigen-driven oligoclonality. Here, we study the B cell repertoire of NOD.H-2h4 mice, a mouse model of spontaneous autoimmunity mimicking SjS without developing diabetes. A library of 168 hybridomas from NOD.H-2h4 mice and 186 C57BL/6J splenocytes at different ages was created. The presence of mono or polyreactive autoantibodies to several antigens was evaluated by ELISA, and their staining patterns and cellular reactivity were tested by IFA and FACS. We observed a higher frequency of autoreactivity among B-cell clones from NOD.H-2h4 mice as compared to wild-type mice. The presence of polyreactive and autoreactive IgG clones increased with mice age. Strikingly, all anti-Ro52 autoantibodies were polyreactive. No loss of polyreactivity was observed upon antibody class switching to IgG. There was a progression to oligoclonality in IgG B cells with mice aging. Our results indicate that in the NOD.H-2h4 mouse model of SjS, IgG+ B cells are mainly polyreactive and might expand following an unknown antigen-driven positive selection process.

Cytomegalovirus restricts ICOSL expression on antigen-presenting cells disabling T cell co-stimulation and contributing to immune evasion.

Viral infections are controlled, and very often cleared, by activated T lymphocytes. The inducible co-stimulator (ICOS) mediates its functions by binding to its ligand ICOSL, enhancing T-cell activation and optimal germinal center (GC) formation. Here, we show that ICOSL is heavily downmodulated during infection of antigen-presenting cells by different herpesviruses. We found that, in murine cytomegalovirus (MCMV), the immunoevasin m138/fcr-1 physically interacts with ICOSL, impeding its maturation and promoting its lysosomal degradation. This viral protein counteracts T-cell responses, in an ICOS-dependent manner, and limits virus control during the acute MCMV infection. Additionally, we report that blockade of ICOSL in MCMV-infected mice critically regulates the production of MCMV-specific antibodies due to a reduction of T follicular helper and GC B cells. Altogether, these findings reveal a novel mechanism evolved by MCMV to counteract adaptive immune surveillance, and demonstrates a role of the ICOS:ICOSL axis in the host defense against herpesviruses.

Efficient elimination of primary B-ALL cells in vitro and in vivo using a novel 4-1BB-based CAR targeting a membrane-distal CD22 epitope.

BACKGROUND: There are few therapeutic options available for patients with B-cell acute lymphoblastic leukemia (B-ALL) relapsing as CD19- either after chemotherapy or CD19-targeted immunotherapies. CD22-chimeric antigen receptor (CAR) T cells represent an attractive addition to CD19-CAR T cell therapy because they will target both CD22+CD19- B-ALL relapses and CD19- preleukemic cells. However, the immune escape mechanisms from CD22-CAR T cells, and the potential contribution of the epitope binding of the anti-CD22 single-chain variable fragment (scFv) remain understudied. METHODS: Here, we have developed and comprehensively characterized a novel CD22-CAR (clone hCD22.7) targeting a membrane-distal CD22 epitope and tested its cytotoxic effects against B-ALL cells both in in vitro and in vivo assays. RESULTS: Conformational epitope mapping, cross-blocking, and molecular docking assays revealed that the hCD22.7 scFv is a high-affinity binding antibody which specifically binds to the ESTKDGKVP sequence, located in the Ig-like V-type domain, the most distal domain of CD22. We observed efficient killing of B-ALL cells in vitro, although the kinetics were dependent on the level of CD22 expression. Importantly, we show an efficient in vivo control of patients with B-ALL derived xenografts with diverse aggressiveness, coupled to long-term hCD22.7-CAR T cell persistence. Remaining leukemic cells at sacrifice maintained full expression of CD22, ruling out CAR pressure-mediated antigen loss. Finally, the immunogenicity capacity of this hCD22.7-scFv was very similar to that of other CD22 scFv previously used in adoptive T cell therapy. CONCLUSIONS: We report a novel, high-affinity hCD22.7 scFv which targets a membrane-distal epitope of CD22. 4-1BB-based hCD22.7-CAR T cells efficiently eliminate clinically relevant B- CD22high and CD22low ALL primary samples in vitro and in vivo. Our study supports the clinical translation of this hCD22.7-CAR as either single or tandem CD22-CD19-CAR for both naive and anti-CD19-resistant patients with B-ALL.

Divergent Traits and Ligand-Binding Properties of the Cytomegalovirus CD48 Gene Family.

The genesis of gene families by the capture of host genes and their subsequent duplication is a crucial process in the evolution of large DNA viruses. CD48 is a cell surface molecule that interacts via its N-terminal immunoglobulin (Ig) domain with the cell surface receptor 2B4 (CD244), regulating leukocyte cytotoxicity. We previously reported the presence of five CD48 homologs (vCD48s) in two related cytomegaloviruses, and demonstrated that one of them, A43, binds 2B4 and acts as a soluble CD48 decoy receptor impairing NK cell function. Here, we have characterized the rest of these vCD48s. We show that they are highly glycosylated proteins that display remarkably distinct features: divergent biochemical properties, cellular locations, and temporal expression kinetics. In contrast to A43, none of them interacts with 2B4. Consistent with this, molecular modeling of the N-terminal Ig domains of these vCD48s evidences notable changes as compared to CD48, suggesting that they interact with alternative targets. Accordingly, we demonstrate that one of them, S30, tightly binds CD2, a crucial T- and NK-cell adhesion and costimulatory molecule. Thus, our findings show how a key host immune receptor gene captured by a virus can be subsequently remodeled to evolve new immunoevasins with altered binding properties.

Cytomegalovirus protein m154 perturbs the adaptor protein-1 compartment mediating broad-spectrum immune evasion.

Cytomegaloviruses (CMVs) are ubiquitous pathogens known to employ numerous immunoevasive strategies that significantly impair the ability of the immune system to eliminate the infected cells. Here, we report that the single mouse CMV (MCMV) protein, m154, downregulates multiple surface molecules involved in the activation and costimulation of the immune cells. We demonstrate that m154 uses its cytoplasmic tail motif, DD, to interfere with the adaptor protein-1 (AP-1) complex, implicated in intracellular protein sorting and packaging. As a consequence of the perturbed AP-1 sorting, m154 promotes lysosomal degradation of several proteins involved in T cell costimulation, thus impairing virus-specific CD8+ T cell response and virus control in vivo. Additionally, we show that HCMV infection similarly interferes with the AP-1 complex. Altogether, we identify the robust mechanism employed by single viral immunomodulatory protein targeting a broad spectrum of cell surface molecules involved in the antiviral immune response.