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V-domain immunoglobulin suppressor of T cell activation (VISTA) is a recently discovered immune checkpoint ligand that functions to suppress T cell activity. The therapeutic potential of activating this immune checkpoint pathway to reduce inflammatory responses remains untapped,largely due to the inability to derive agonists targeting its unknown receptor. A dimeric construct of the IgV domain of VISTA (VISTA-Fc) was shown to suppress the activation of T cells in vitro. However,this effect required its immobilization on a solid surface,suggesting that VISTA-Fc may display limited efficacy as a VISTA-receptor agonist in vivo. Herein,we have designed a stable pentameric VISTA construct (VISTA.COMP) by genetically fusing its IgV domain to the pentamerization domain from the cartilage oligomeric matrix protein (COMP). In contrast to VISTA-Fc,VISTA.COMP does not require immobilization to inhibit the proliferation of CD4+ T cells undergoing polyclonal activation. Furthermore,we show that VISTA.COMP,but not VISTA-Fc,functions as an immunosuppressive agonist in vivo capable of prolonging the survival of skin allografts in a mouse transplant model as well as rescuing mice from acute concanavalin-A-induced hepatitis. Collectively,we believe our data demonstrate that VISTA.COMP is a checkpoint receptor agonist and the first agent to our knowledge targeting the putative VISTA-receptor to suppress T cell-mediated immune responses. View Publication

Diverse hematopoietic progenitors,including myeloid populations arising in inflammatory and tumoral conditions and multipotent cells,mobilized by hematopoietic growth factors or emerging during parasitic infections,display tolerogenic properties. Innate immune stimuli confer regulatory functions to various mature B-cell subsets but immature B-cell progenitors endowed with suppressive properties per se or after differentiating into more mature regulatory B cells remain to be characterized. Herein we provide evidence for innate pro-B cells (CpG-proBs) that emerged within the bone marrow both in vitro and in vivo upon Toll-like receptor-9 activation and whose adoptive transfer protected nonobese diabetic mice against type 1 diabetes (T1D). These cells responded to IFN-$\gamma$ released by activated effector T cells (Teffs),by up-regulating their Fas ligand (FasL) expression,which enabled them to kill Teffs through apoptosis. In turn,IFN-$\gamma$ derived from CpG-proBs enhanced IFN-$\gamma$ while dramatically reducing IL-21 production by Teffs. In keeping with the crucial pathogenic role played by IL-21 in T1D,adoptively transferred IFN-$\gamma$-deficient CpG-proBs did not prevent T1D development. Additionally,CpG-proBs matured in vivo into diverse pancreatic and splenic suppressive FasL(high) B-cell subsets. CpG-proBs may become instrumental in cell therapy of autoimmune diseases either on their own or as graft complement in autologous stem cell transplantation. View Publication

Type 1 diabetes (T1D) is an autoimmune disease in which a strong inflammatory response causes the death of insulin-producing pancreatic beta-cells,while inefficient regulatory mechanisms allow that response to become chronic. Ethyl pyruvate (EP),a stable pyruvate derivate and certified inhibitor of an alarmin-high mobility group box 1 (HMGB1),exerts anti-oxidant and anti-inflammatory properties in animal models of rheumatoid arthritis and encephalomyelitis. To test its therapeutic potential in T1D,EP was administered intraperitoneally to C57BL/6 mice with multiple low-dose streptozotocin (MLDS)-induced T1D. EP treatment decreased T1D incidence,reduced the infiltration of cells into the pancreatic islets and preserved beta-cell function. Apart from reducing HMGB1 expression,EP treatment successfully interfered with the inflammatory response within the local pancreatic lymph nodes and in the pancreas. Its effect was restricted to boosting the regulatory arm of the immune response through up-regulation of tolerogenic dendritic cells (CD11c+CD11b-CD103+) within the pancreatic infiltrates and through the enhancement of regulatory T cell (Treg) levels (CD4+CD25highFoxP3+). These EP-stimulated Treg displayed enhanced suppressive capacity reflected in increased levels of CTLA-4,secreted TGF-beta,and IL-10 and in the more efficient inhibition of effector T cell proliferation compared to Treg from diabetic animals. Higher levels of Treg were a result of increased differentiation and proliferation (Ki67+ cells),but also of the heightened potency for migration due to increased expression of adhesion molecules (CD11a and CD62L) and CXCR3 chemokine receptor. Treg isolated from EP-treated mice had the activated phenotype and T-bet expression more frequently,suggesting that they readily suppressed IFN-gamma-producing cells. The effect of EP on Treg was also reproduced in vitro. Overall,our results show that EP treatment reduced T1D incidence in C57BL/6 mice predominantly by enhancing Treg differentiation,proliferation,their suppressive capacity,and recruitment into the pancreas. View Publication

Tissue-repair regulatory T cells (trTregs) comprise a specialized cell subset essential for tissue homeostasis and repair. While well-studied in sterile injury models,their role in infection-induced tissue damage and antimicrobial immunity is less understood. We investigated trTreg dynamics during acute Trypanosoma cruzi infection,marked by extensive tissue damage and strong CD8+ immunity. Unlike sterile injury models,trTregs significantly declined in secondary lymphoid organs and non-lymphoid target tissues during infection,correlating with systemic and local tissue damage,and downregulation of function-associated genes in skeletal muscle. This decline was linked to decreased systemic IL-33 levels,a key trTreg growth factor,and promoted by the Th1 cytokine IFN-γ. Early recombinant IL-33 treatment increased trTregs,type 2 innate lymphoid cells,and parasite-specific CD8+ cells at specific time points after infection,leading to reduced tissue damage,lower parasite burden,and improved disease outcome. Our findings not only provide novel insights into trTregs during infection but also highlight the potential of optimizing immune balance by modulating trTreg responses to promote tissue repair while maintaining effective pathogen control during infection-induced injury. Author summaryDuring Chagas’ disease,caused by the protozoan Trypanosoma cruzi,severe organ damage is generated by the interplay between the parasite and the immune response. In our investigation,we examined the role of tissue-repair regulatory T cells (trTregs) during the acute phase of T. cruzi infection in mice. Surprisingly,we observed a reduction in trTregs at the peak of tissue damage,contrary to their usual accumulation after injury in other contexts. This decline aligned with decreased levels of interleukin-33,a critical factor for trTreg survival,and was promoted by the effector cytokine IFN-γ. Administering interleukin-33 at early infection times not only boosted trTregs but also expanded other reparative and antiparasitic immune cells. Consequently,these treated mice exhibited reduced damage and lower parasite levels in tissues. Our findings provide new insights into how trTreg function during infection-related injury,paving the way for strategies that balance the immune response to support tissue repair without weakening the body’s ability to fight the infection. This approach could have broader implications for treating infectious diseases and conditions involving tissue damage. View Publication

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by anti-nuclear autoantibodies whose production is promoted by autoreactive T follicular helper (TFH) cells. During SLE pathogenesis,basophils accumulate in secondary lymphoid organs (SLO),amplify autoantibody production and disease progression through mechanisms that remain to be defined. Here,we provide evidence for a direct functional relationship between TFH cells and basophils during lupus pathogenesis,both in humans and mice. PD-L1 upregulation on basophils and IL-4 production are associated with TFH and TFH2 cell expansions and with disease activity. Pathogenic TFH cell accumulation,maintenance,and function in SLO were dependent on PD-L1 and IL-4 in basophils,which induced a transcriptional program allowing TFH2 cell differentiation and function. Our study establishes a direct mechanistic link between basophils and TFH cells in SLE that promotes autoantibody production and lupus nephritis. Basophils have been implicated in systemic lupus erythematosus (SLE),as evidenced by the fact that basophil-deficient mice do not develop the disease. Here,the authors demonstrate that PD-L1 and IL-4 expression in basophils promotes the pathogenic accumulation of follicular helper T cells in patients with SLE and murine models. View Publication

Lymphatic endothelial cells (LECs) chemoattract na{\{i}}ve T cells and promote their survival in the lymph nodes and can cross-present antigens to na{\"{i}}ve CD8+ T cells to drive their proliferation despite lacking key costimulatory molecules. However the functional consequence of LEC priming of CD8+ T cells is unknown. Here we show that while many proliferating LEC-educated T cells enter early apoptosis the remainders comprise a long-lived memory subset with transcriptional metabolic and phenotypic features of central memory and stem cell-like memory T cells. In vivo these memory cells preferentially home to lymph nodes and display rapid proliferation and effector differentiation following memory recall and can protect mice against a subsequent bacterial infection. These findings introduce a new immunomodulatory role for LECs in directly generating a memory-like subset of quiescent yet antigen-experienced CD8+ T cells that are long-lived and can rapidly differentiate into effector cells upon inflammatory antigenic challenge.""" View Publication

The active movement of cells from subendothelial compartments into the bloodstream (intravasation) has been recognized for several decades by histologic and physiologic studies,yet the molecular effectors of this process are relatively uncharacterized. For extravasation,studies based predominantly on static transwell assays support a general model,whereby transendothelial migration (TEM) occurs via chemoattraction toward increasing chemokine concentrations. However,this model of chemotaxis cannot readily reconcile how chemokines influence intravasation,as shear forces of blood flow would likely abrogate luminal chemokine gradient(s). Thus,to analyze how T cells integrate perivascular chemokine signals under physiologic flow,we developed a novel transwell-based flow chamber allowing for real-time modulation of chemokine levels above (luminal/apical compartment) and below (abluminal/subendothelial compartment) HUVEC monolayers. We routinely observed human T cell TEM across HUVEC monolayers with the combination of luminal CXCL12 and abluminal CCL5. With increasing concentrations of CXCL12 in the luminal compartment,transmigrated T cells did not undergo retrograde transendothelial migration (retro-TEM). However,when exposedto abluminal CXCL12,transmigrated T cells underwent striking retro-TEM and re-entered the flow stream [corrected]. This CXCL12 fugetactic (chemorepellant) effect was concentration-dependent,augmented by apical flow,blocked by antibodies to integrins,and reduced by AMD3100 in a dose-dependent manner. Moreover,CXCL12-induced retro-TEM was inhibited by PI3K antagonism and cAMP agonism. These findings broaden our understanding of chemokine biology and support a novel paradigm by which temporospatial modulations in subendothelial chemokine display drive cell migration from interstitial compartments into the bloodstream. View Publication

MICA is a stress-regulated molecule recognized by the NK cell-activating receptor NKG2D. Previously,we demonstrated that MICA is induced on activated T cells but regulation by mitogenic cytokines and its biological consequences remain unexplored. Here,we show that IL-2,IL-4,and IL-15 but not TNF-alpha or IFN-alpha induced MICA expression in T lymphocytes present in peripheral blood mononuclear cells (PBMCs),as assessed by Western blot. IL-2 effect involved Jak3/STAT5,p38 MAPK,p70(56) kinase,Lck/fyn kinases,and NF-kappaB. MICA expression was also observed in Th1 and Th2 cells. However,surface expression was not detected. T lymphocytes present in PBMCs and isolated CD4+ T lymphocytes stimulated with phorbol-12-myristate-13-acetate and ionomycin also induced MICA expression as assessed by Western blot,but only low levels were expressed at the cell surface. Activated but not resting CD4+ T lymphocytes were lysed by IL-15- or IL-2-stimulated NK cells,and susceptibility was increased when HLA class I molecules were blocked. Also,cytokine-stimulated NK cells produced more IFN-gamma after culture with activated CD4+ T lymphocytes. However,the participation of MICA in these responses,if any,was marginal. Confocal microscopy revealed that MICA is retained mostly inside activated CD4+ T cells. Our results suggest that low surface expression of MICA on activated CD4+ T lymphocytes might be a safeguard mechanism to protect them from NK cells in an inflammatory,virus-infected,or tumor microenvironment,where NK and activated CD4+ T cells are recruited. View Publication

Cancer-initiating cells (CICs) that are responsible for tumor initiation,propagation,and resistance to standard therapies have been isolated from human solid tumors,including colorectal cancer (CRC). The aim of this study was to obtain an immunological profile of CRC-derived CICs and to identify CIC-associated target molecules for T cell immunotherapy. We have isolated cells with CIC properties along with their putative non-CIC autologous counterparts from human primary CRC tissues. These CICs have been shown to display tumor-initiating/stemness" properties� View Publication

Human pluripotent stem cells will likely be a significant part of the regenerative medicine-driven healthcare revolution. In order to realize this potential,culture processes must be standardized,scalable and able to produce clinically relevant cell numbers,whilst maintaining critical biological functionality. This review comprises a broad overview of important bioprocess considerations,referencing the development of biopharmaceutical processes in an effort to learn from current best practice in the field. Particular focus is given to the recent efforts to grow human pluripotent stem cells in microcarrier or aggregate suspension culture,which would allow geometric expansion of productive capacity were it to be fully realized. The potential of these approaches is compared with automation of traditional T-flask culture,which may provide a cost-effective platform for low-dose,low-incidence conditions or autologous therapies. This represents the first step in defining the full extent of the challenges facing bioprocess engineers in the exploitation of large-scale human pluripotent stem cell manufacture. View Publication

Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent antitumor effects in B-cell malignancies including acute lymphoblastic leukemia (ALL),but antigen loss remains the major cause of treatment failure. To mitigate antigen escape and potentially improve the durability of remission,we developed a dual-targeting approach using an optimized,bispecific CAR construct that targets both CD19 and BAFF-R. CD19/BAFF-R dual CAR T cells exhibited antigen-specific cytokine release,degranulation,and cytotoxicity against both CD19-/- and BAFF-R-/- variant human ALL cells in vitro. Immunodeficient mice engrafted with mixed CD19-/- and BAFF-R-/- variant ALL cells and treated with a single dose of CD19/BAFF-R dual CAR T cells experienced complete eradication of both CD19-/- and BAFF-R-/- ALL variants,whereas mice treated with monospecific CD19 or BAFF-R CAR T cells succumbed to outgrowths of CD19-/BAFF-R+ or CD19+/BAFF-R- tumors,respectively. Further,CD19/BAFF-R dual CAR T cells showed prolonged in vivo persistence,raising the possibility that these cells may have the potential to promote durable remissions. Together,our data support clinical translation of BAFF-R/CD19 dual CAR T cells to treat ALL. View Publication

Adult T-cell leukemia/lymphoma (ATL) occurs after human T-cell leukemia virus type-1 (HTLV-1) infection with a long latency period exceeding several decades. This implies the presence of immune evasion mechanisms for HTLV-1-infected T cells. Although ATL cells have a CD4 + CD25 + phenotype similar to that of regulatory T cells (Tregs),they do not always possess the immunosuppressive functions of Tregs. Factors that impart effective immunosuppressive functions to HTLV-1-infected cells may exist. A previous study identified a new CD13 + Treg subpopulation with enhanced immunosuppressive activity. We,herein,describe the paired CD13 − (designated as MT-50.1) and CD13 + (MT-50.4) HTLV-1-infected T-cell lines with Treg-like phenotype,derived from the peripheral blood of a single patient with lymphoma-type ATL. The cell lines were found to be derived from HTLV-1-infected non-leukemic cells. MT-50.4 cells secreted higher levels of immunosuppressive cytokines,IL-10 and TGF-β,expressed higher levels of Foxp3,and showed stronger suppression of CD4 + CD25 − T cell proliferation than MT-50.1 cells. Furthermore,the CD13 inhibitor bestatin significantly attenuated MT-50.4 cell growth,while it did not for MT-50.1 cells. These findings suggest that CD13 expression may be involved in the increased Treg-like activity of MT-50.4 cells. Hence,MT-50.4 cells will be useful for in-depth studies of CD13 + Foxp3 + HTLV-1-infected cells. Subject terms: Cancer,Microbiology,Oncology View Publication