Scientific Resources

Purinergic signaling plays a major role in T cell activation leading to IL-2 production and proliferation. However,it is unclear whether purinergic signaling contributes to the differentiation and activation of effector T cells. In this study,we found that the purinergic receptor P2X4 was associated with human Th17 cells but not with Th1 cells. Inhibition of P2X4 receptor with the specific antagonist 5-BDBD and small interfering RNA inhibited the development of Th17 cells and the production of IL-17 by effector Th17 cells stimulated via the CD3/CD28 pathway. Our results showed that P2X4 was required for the expression of retinoic acid-related orphan receptor C,which is the master regulator of Th17 cells. In contrast,inhibition of P2X4 receptor had no effect on Th1 cells and on the production of IFN-? and it did not affect the expression of the transcription factor T-bet (T-box transcription factor). Furthermore,inhibition of P2X4 receptor reduced the production of IL-17 but not of IFN-? by effector/memory CD4+ T cells isolated from patients with rheumatoid arthritis. In contrast to P2X4,inhibition of P2X7 and P2Y11 receptors had no effects on Th17 and Th1 cell activation. Finally,treatment with the P2X4 receptor antagonist 5-BDBD reduced the severity of collagen-induced arthritis in mice by inhibiting Th17 cell expansion and activation. Our findings provide novel insights into the role of purinergic signaling in T cell activation and identify a critical role for the purinergic receptor P2X4 in Th17 activation and in autoimmune arthritis. View Publication

Rationale: T cell therapeutic strategy using CD19-targeting chimeric antigen receptor (CAR) is a revolutionary,novel,and successful treatment for B-cell malignancies. However,the dependency on T-cell mediated cytotoxicity restricts CAR-T therapy as a patient-specific individualized therapy with severe side effects,such as cytokine release syndrome (CRS). Whether a non-T-cell based universal cellular therapy can substitute CAR-T therapy is largely unknown. Methods: Various artificial antigen-recognizing cells were prepared to determine whether non-T-cell-derived CD19-scFv bearing effector cells could cause target cell death. A universal strategy for CRS-free cellular therapeutics was proposed,utilizing artificial antigen-recognizing cells (AARC),which can be manufactured universally and routinely as off-the-shelf" mesenchymal stromal cells (MSCs) or other types of non-autologous cells expressing anergic CARs. Results: We demonstrated that T-lymphocytic and non-lymphocytic cells could cause CD19 internalization and subsequent depletion when armed with a CD19-recognizing moiety. This CD19 antigen depletion could efficiently induce T-cell independent apoptosis in target cancer cells whose survival depends on CD19 expression suggesting that CD19 antigen depletion constitutes a crucial tumor destroying mechanism for CD19-CAR-T especially for its long-term efficacy. Conclusion: Our results uncovered an unrecognized CAR-T cytotoxicity and antigen loss mechanism and provided new insights into a shift from unique patient-specific autologous therapeutics to universal and standardized allogeneic treatment." View Publication

Heterologous immunity,when the memory T cell response elicited by one pathogen recognizes another pathogen,has been offered as a contributing factor for the high variability in coronavirus disease 2019 (COVID-19) severity outcomes. Here we demonstrate that sensitization with bacterial peptides can induce heterologous immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) derived peptides and that vaccination with the SARS-CoV-2 spike protein can induce heterologous immunity to bacterial peptides. Using in silico prediction methods,we identified 6 bacterial peptides with sequence homology to either the spike protein or non-structural protein 3 (NSP3) of SARS-CoV-2. Notwithstanding the effects of bystander activation,in vitro co-cultures showed that all individuals tested (n=18) developed heterologous immunity to SARS-CoV-2 peptides when sensitized with the identified bacterial peptides. T cell recall responses measured included cytokine production (IFN-$\gamma$,TNF,IL-2),activation (CD69) and proliferation (CellTrace). As an extension of the principle of heterologous immunity between bacterial pathogens and COVID-19,we tracked donor responses before and after SARS-CoV-2 vaccination and measured the cross-reactive T cell responses to bacterial peptides with similar sequence homology to the spike protein. We found that SARS-CoV-2 vaccination could induce heterologous immunity to bacterial peptides. These findings provide a mechanism for heterologous T cell immunity between common bacterial pathogens and SARS-CoV-2,which may explain the high variance in COVID-19 outcomes from asymptomatic to severe. We also demonstrate proof-of-concept that SARS-CoV-2 vaccination can induce heterologous immunity to pathogenic bacteria derived peptides. View Publication

Umbilical cord blood (UCB) is an alternative source of allogeneic hematopoietic stem cells (HSCs) for transplantation to treat various hematological disorders. The major limitation to the use of UCB-derived HSCs (UCB-HSCs) in transplantation,however,is the low numbers of HSCs in a unit of cord blood. To overcome this limitation,various cytokines or small molecules have been used to expand UCB-HSCs ex vivo. In this study,we investigated a synergistic effect of the combination of HIL-6,SR1,and UM171 on UCB-HSC culture and found that this combination resulted in the highest number of CD34+ cells. These results suggest that the combination of SR1,UM171 and HIL-6 exerts a synergistic effect in the proliferation of HSCs from UCB and thus,SR1,UM171 and HIL-6 is the most suitable combination for obtaining HSCs from UCB for clinical transplantation. View Publication

OBJECTIVE True identity and specific set of markers to enrich endometrial stem cells still remains elusive. Present study was undertaken to further substantiate that very small embryonic-like stem cells (VSELs) are the true and elusive stem cells in adult mice endometrium. METHODS This was achieved by undertaking three sets of experiments. Firstly,SSEA-1+ and Oct-4??+??positive VSELs,sorted from GFP mice,were transplanted into the uterine horns of wild-type Swiss mice and GFP uptake was studied within the same estrus cycle. Secondly,uterine lumen was scratched surgically and OCT-4 expressing stem/progenitor cells were studied at the site of injury after 24-72 h. Thirdly,OCT-4????expression was studied in the endometrium and myometrium of adult mice after neonatal exposure to estradiol (20 µg/pup/day on days 5-7 after birth). RESULTS GFP??+??ve VSELs expressing SSEA-1 and Oct-4 engrafted and differentiated into the epithelial cells lining the lumen as well as the glands during the estrus stage when maximum remodeling occurs. Mechanical scratching activated tissue-resident,nuclear OCT-4 positive VSELs and slightly bigger 'progenitors' endometrial stem cells (EnSCs,cytoplasmic OCT-4) which underwent clonal expansion and further differentiated into luminal and glandular epithelial cells. Neonatal exposure to endocrine disruption resulted in increased numbers of OCT-4 positive VSELs/EnSCs in adult endometrium. DISCUSSION Results support the presence of functionally active VSELs in adult endometrium. VSELs self-renew and give rise to EnSCs that further differentiate into epithelial cells under normal physiological conditions. Also,VSELs are vulnerable to endocrine insults. To conclude VSELs are true and elusive uterine stem cells that maintain life-long uterine homeostasis and their dysregulation may result in various pathologies. View Publication

Cx3cr1+ monocyte-derived macrophages (moMacs) are recruited to tissues after injury and are known to have profibrotic effects,but the cell-cell interactions and specific pathways that regulate this polarization and function are incompletely understood. Here we investigate the role of moMac-derived Pdgfa in bleomycin-induced lung fibrosis in mice. Deletion of Pdgfa with Cx3cr1-CreERT2 decreased bleomycin-induced lung fibrosis. Among a panel of in vitro macrophage polarizing stimuli,robust induction of Pdgfa was noted with IL10 in both mouse and human moMacs. Likewise,analysis of single-cell data revealed high expression of the receptor IL10RA in moMacs from human fibrotic lungs. Studies with IL10-GFP mice revealed that IL10-expressing cells were increased after injury in mice and colocalized with moMacs. Notably,deletion of IL10ra with Csf1r-Cre: IL10ra fl/fl mice decreased both Pdgfa expression in moMacs and lung fibrosis. Taken together,these findings reveal a novel,IL10-dependent mechanism of macrophage polarization leading to fibroblast activation after injury. View Publication

Dysregulated chronic inflammation plays a crucial role in the pathophysiology of atherosclerosis and may be a result of impaired resolution. Thus,restoring levels of specialized pro-resolving mediators (SPMs) to promote the resolution of inflammation has been proposed as a therapeutic strategy for patients with atherosclerosis,in addition to standard clinical care. Herein,we evaluated the effects of the SPM lipids,lipoxin A4 (LXA4 ) and lipoxin B4 (LXB4 ),on neutrophils isolated from patients with atherosclerosis compared with healthy controls. Patients displayed altered endogenous SPM production,and we demonstrated that lipoxin treatment in whole blood from atherosclerosis patients attenuates neutrophil oxidative burst,a key contributor to atherosclerotic development. We found the opposite effect in neutrophils from healthy controls,indicating a potential mechanism whereby lipoxins aid the endogenous neutrophil function in health but reduce its excessive activation in disease. We also demonstrated that lipoxins attenuated upregulation of the high-affinity conformation of the CD11b/CD18 integrin,which plays a central role in clot activation and atherosclerosis. Finally,LXB4 enhanced lymphatic transmigration of human neutrophils isolated from patients with atherosclerosis. This finding is noteworthy,as impaired lymphatic function is now recognized as an important contributor to atherosclerosis. Although both lipoxins modulated neutrophil function,LXB4 displayed more potent effects than LXA4 in humans. This study highlights the therapeutic potential of lipoxins in atherosclerotic disease and demonstrates that the effect of these SPMs may be specifically tailored to the need of the individual. View Publication

CD8+ T cell responses are the foundation of the recent clinical success of immunotherapy in oncologic indications. Although checkpoint inhibitors have enhanced the activity of existing CD8+ T cell responses,therapeutic approaches to generate Ag-specific CD8+ T cell responses have had limited success. Here,we demonstrate that cytosolic delivery of Ag through microfluidic squeezing enables MHC class I presentation to CD8+ T cells by diverse cell types. In murine dendritic cells (DCs),squeezed DCs were ˆ¼1000-fold more potent at eliciting CD8+ T cell responses than DCs cross-presenting the same amount of protein Ag. The approach also enabled engineering of less conventional APCs,such as T cells,for effective priming of CD8+ T cells in vitro and in vivo. Mixtures of immune cells,such as murine splenocytes,also elicited CD8+ T cell responses in vivo when squeezed with Ag. We demonstrate that squeezing enables effective MHC class I presentation by human DCs,T cells,B cells,and PBMCs and that,in clinical scale formats,the system can squeeze up to 2 billion cells per minute. Using the human papillomavirus 16 (HPV16) murine model,TC-1,we demonstrate that squeezed B cells,T cells,and unfractionated splenocytes elicit antitumor immunity and correlate with an influx of HPV-specific CD8+ T cells such that >80% of CD8s in the tumor were HPV specific. Together,these findings demonstrate the potential of cytosolic Ag delivery to drive robust CD8+ T cell responses and illustrate the potential for an autologous cell-based vaccine with minimal turnaround time for patients. View Publication

Pivotal clinical trials of B-cell maturation antigen-targeted chimeric antigen receptor T (CART)-cell therapy in patients with relapsed/refractory multiple myeloma (MM) resulted in remarkable initial responses,which led to a recent US Food and Drug Administration approval. Despite the success of this therapy,durable remissions continue to be low,and the predominant mechanism of resistance is loss of CART cells and inhibition by the tumor microenvironment (TME). MM is characterized by an immunosuppressive TME with an abundance of cancer-associated fibroblasts (CAFs). Using MM models,we studied the impact of CAFs on CART-cell efficacy and developed strategies to overcome CART-cell inhibition. We showed that CAFs inhibit CART-cell antitumor activity and promote MM progression. CAFs express molecules such as fibroblast activation protein and signaling lymphocyte activation molecule family-7,which are attractive immunotherapy targets. To overcome CAF-induced CART-cell inhibition,CART cells were generated targeting both MM cells and CAFs. This dual-targeting CART-cell strategy significantly improved the effector functions of CART cells. We show for the first time that dual targeting of both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART-cell therapy in MM. View Publication

Sialic acids are negatively charged carbohydrates that cap the glycans of glycoproteins and glycolipids. Sialic acids are involved in various biological processes including cell-cell adhesion and immune recognition. In dendritic cells (DCs),the major antigen-presenting cells of the immune system,sialic acids emerge as important regulators of maturation and interaction with other lymphocytes including T cells. Many aspects of how sialic acids regulate DC functions are not well understood and tools and model systems to address these are limited. Here,we have established cultures of murine bone marrow-derived DCs (BMDCs) that lack sialic acid expression using a sialic acid-blocking mimetic Ac53FaxNeu5Ac. Ac53FaxNeu5Ac treatment potentiated BMDC activation via toll-like receptor (TLR) stimulation without affecting differentiation and viability. Sialic acid blockade further increased the capacity of BMDCs to induce antigen-specific CD8+ T cell proliferation. Transcriptome-wide gene expression analysis revealed that sialic acid mimetic treatment of BMDCs induces differential expression of genes involved in T cell activation,cell-adhesion,and cell-cell interactions. Subsequent cell clustering assays and single cell avidity measurements demonstrated that BMDCs with reduced sialylation form higher avidity interactions with CD8+ T cells. This increased avidity was detectable in the absence of antigens,but was especially pronounced in antigen-dependent interactions. Together,our data show that sialic acid blockade in BMDCs ameliorates maturation and enhances both cognate T cell receptor-MHC-dependent and independent T cell interactions that allow for more robust CD8+ T cell responses. View Publication

A subset of antibodies found in cattle comprises ultralong CDR-H3 regions of up to 70 amino acids. Interestingly,this type of immunoglobulin usually pairs with the single germline VL gene,V30 that is typically very conserved in sequence. In this work,we have engineered ultralong CDR-H3 common light chain bispecific antibodies targeting Epidermal Growth Factor Receptor (EGFR) on tumor cells as well as Natural Cytotoxicity Receptor NKp30 on Natural Killer (NK) cells. Antigen-specific common light chain antibodies were isolated by yeast surface display by means of pairing CDR-H3 diversities following immunization with a single V30 light chain. After selection,EGFR-targeting paratopes as well as NKp30-specific binders were combined into common light chain bispecific antibodies by exploiting the strand-exchange engineered domain (SEED) technology for heavy chain heterodimerization. Biochemical characterization of resulting bispecifics revealed highly specific binding to the respective antigens as well as simultaneous binding to both targets. Most importantly,engineered cattle-derived bispecific common light chain molecules elicited potent NK cell redirection and consequently tumor cell lysis of EGFR-overexpressing cells as well as robust release of proinflammatory cytokine interferon-$\gamma$. Taken together,this data is giving clear evidence that bovine bispecific ultralong CDR-H3 common light chain antibodies are versatile for biotechnological applications. View Publication

Solid tumor cells have an altered metabolism that can protect them from cytotoxic lymphocytes. The anti-diabetic drug metformin modifies tumor cell metabolism and several clinical trials are testing its effectiveness for the treatment of solid cancers. The use of metformin in hematologic cancers has received much less attention,although allogeneic cytotoxic lymphocytes are very effective against these tumors. We show here that metformin induces expression of Natural Killer G2-D (NKG2D) ligands (NKG2DL) and intercellular adhesion molecule-1 (ICAM-1),a ligand of the lymphocyte function-associated antigen 1 (LFA-1). This leads to enhance sensitivity to cytotoxic lymphocytes. Overexpression of anti-apoptotic Bcl-2 family members decrease both metformin effects. The sensitization to activated cytotoxic lymphocytes is mainly mediated by the increase on ICAM-1 levels,which favors cytotoxic lymphocytes binding to tumor cells. Finally,metformin decreases the growth of human hematological tumor cells in xenograft models,mainly in presence of monoclonal antibodies that recognize tumor antigens. Our results suggest that metformin could improve cytotoxic lymphocyte-mediated therapy. View Publication