Following antigen stimulation, naïve T cells differentiate into memory cells that mediate antigen clearance more efficiently upon repeat encounter. Donor-specific tolerance can be achieved in a subset of transplant recipients, but some of these grafts are rejected after years of stability, often following infections. Whether T cell memory can develop from a tolerant state and whether these formerly tolerant patients develop antidonor memory is not known. Using a mouse model of cardiac transplantation in which donor-specific tolerance is induced with costimulation blockade (CoB) plus donor-specific transfusion (DST), we have previously shown that systemic infection with Listeria monocytogenes (Lm) months after transplantation can erode or transiently abrogate established tolerance. In this study, we tracked donor-reactive T cells to investigate whether memory can be induced when alloreactive T cells are activated in the setting of tolerance. We show alloreactive T cells persist after induction of cardiac transplantation tolerance, but fail to acquire a memory phenotype despite becoming antigen experienced. Instead, donor-reactive T cells develop T cell-intrinsic dysfunction evidenced when removed from the tolerant environment. Notably, Lm infection after tolerance did not rescue alloreactive T cell memory differentiation or functionality. CoB and antigen persistence were sufficient together but not separately to achieve alloreactive T cell dysfunction, and conventional immunosuppression could substitute for CoB. Antigen persistence was required, as early but not late surgical allograft removal precluded the acquisition of T cell dysfunction. Our results demonstrate transplant tolerance-associated T cell-intrinsic dysfunction that is resistant to memory development even after Lm-mediated disruption of tolerance.

Recent endeavors to use stem cells as trophic factor production sources have the potential to translate into viable therapies for damaged or diseased musculoskeletal tissues. Adipose stem cells (ASCs) can be differentiated into chondrocytes using the chondrogenic medium (CM), but it is unknown if this approach can optimize ASC growth factor secretion for cartilage regeneration by increasing the chondrogenic factor production, while decreasing angiogenic and hypertrophic factor production. The objective of this study was to determine the effects the CM and its components have on growth factor production from ASCs to promote cartilage regeneration. ASCs isolated from male Sprague-Dawley rats and cultured in monolayer or alginate microbeads were treated with either the growth medium (GM) or the CM for 5 days. In subsequent studies, ASC monolayers were treated with either the GM supplemented with different combinations of 50 μg/mL ascorbic acid-2-phosphate (AA2P), 100 nM dexamethasone (Dex), 10 ng/mL transforming growth factor (TGF)-β1, and 100 ng/mL bone morphogenetic protein (BMP)-6 or with the CM excluding different combinations of AA2P, Dex, TGF-β1, and BMP-6. mRNA levels and growth factor production were quantified at 8 and 24 h after the last media change, respectively. The CM increased chondrogenic factor secretion (TGF-β2, TGF-β3, and insulin-like growth factor [IGF]-I) and decreased angiogenic factor production (the vascular endothelial growth factor [VEGF]-A, the fibroblast growth factor [FGF]-2). Microencapsulation in the GM increased production of the chondrogenic (IGF-I, TGF-β2) and angiogenic (VEGF-A) factors. AA2P increased secretion of chondrogenic factors (IGF-I, TGF-β2), and decreased angiogenic factor (VEGF-A) secretion, in addition to decreasing mRNA levels for factors associated with chondrocyte hypertrophy (FGF-18). Dex increased mRNA levels for hypertrophic factors (BMP-2, FGF-18) and decreased angiogenic factor secretion (VEGF-A). TGF-β1 increased angiogenic factor production (FGF-2, VEGF-A) and decreased chondrogenic factor mRNA levels (IGF-I, PTHrP). BMP-6 increased hypertrophic mRNA levels (FGF-18) and chondrogenic factor production (TGF-β2). When ASC microbeads preconditioned with the CM were implanted in a focal cartilage defect and immobilized within an RGD-conjugated hydrogel, tissue infiltration from the edges of the defect and perichondrium was observed. These results show that differentiation media components have distinct effects on ASC's production of angiogenic, chondrogenic, and hypertrophic factors and that AA2P may be the most beneficial CM component for preconditioning ASCs to stimulate cartilage regeneration.

Inverse vaccines that tolerogenically target antigens to antigen-presenting cells (APCs) offer promise in prevention of immunity to allergens and protein drugs and treatment of autoimmunity. We have previously shown that targeting hepatic APCs through intravenous injection of synthetically glycosylated antigen leads to effective induction of antigen-specific immunological tolerance. Here, we demonstrate that targeting these glycoconjugates to lymph node (LN) APCs under homeostatic conditions leads to local and increased accumulation in the LNs compared to unmodified antigen and induces a tolerogenic state both locally and systemically. Subcutaneous administration directs the polymeric glycoconjugate to the draining LN, where the glycoconjugated antigen generates robust antigen-specific CD4+ and CD8+ T cell tolerance and hypo-responsiveness to antigenic challenge via a number of mechanisms, including clonal deletion, anergy of activated T cells, and expansion of regulatory T cells. Lag-3 up-regulation on CD4+ and CD8+ T cells represents an essential mechanism of suppression. Additionally, presentation of antigen released from the glycoconjugate to naïve T cells is mediated mainly by LN-resident CD8+ and CD11b+ dendritic cells. Thus, here we demonstrate that antigen targeting via synthetic glycosylation to impart affinity for APC scavenger receptors generates tolerance when LN dendritic cells are the cellular target.

The revolutionizing efficacy of recombinant human bone morphogenetic protein (rhBMP-2) for clinical spinal fusion is hindered by safety issues associated with the high dose required. However, it continues to be widely used, for example, in InFUSE Bone Graft (Medtronic). Here, we developed a translational protein engineering–based approach to reduce the dose and thereby improve the safety of rhBMP-2 delivered in a collagen sponge, as in InFUSE Bone Graft. We engineered a bridge protein with high affinity for rhBMP-2 and collagen that can be simply added to the product’s formulation, demonstrating improved efficacy at low dose of rhBMP-2 in two mouse models of bone regeneration, including a newly developed spinal fusion model. Moreover, the bridge protein can control the retention of rhBMP-2 from endogenous collagenous extracellular matrix of tissue. Our approach may be generalizable to other growth factors and collagen-based materials, for use in many other applications in regenerative medicine.

Interleukin-4 (IL-4) suppresses the development of multiple sclerosis in a murine model of experimental autoimmune encephalomyelitis (EAE). Here, we show in mice with EAE that, compared with the administration of wild-type IL-4 or of the clinically approved drug fingolimod, the systemic injection of serum albumin (SA) fused to IL-4 better accumulates and persists in lymph nodes and in the spleen, leading to higher therapeutic efficacy and to the prevention of disease development in the majority of the mice. We also show that the SA-IL-4 fusion protein prevented immune-cell infiltration in the spinal cord, decreased integrin expression in antigen-specific CD4 + T cells, increased the number of granulocyte-like myeloid-derived suppressor cells (and their expression of programmed-death-ligand-1) in spinal-cord-draining lymph nodes, and decreased the number of T helper 17 cells, a pathogenic cell population in EAE. In mice with chronic EAE, SA-IL-4 inhibited immune-cell infiltration into the spinal cord and completely abrogated immune responses to myelin antigen in the spleen. The SA-IL-4 fusion protein may be prophylactically and therapeutically advantageous in the treatment of multiple sclerosis. Multiple sclerosis (MS) is a potentially disabling autoimmune disease that affects millions globally. Autoreactive immune cells home to the central nervous system (CNS) and cause demyelination and consequently focal damage to white matter 1 . Lymphocytes and macrophages that have infiltrated into the CNS cause axonal damage. Recent studies have shown that Th17 cells, activated in the secondary lymphoid organs (SLOs), migrate to the spinal cord and brain and play a crucial role in the disease development and severity of MS 2, 3 . Thus, inhibition of lymphocyte migration to the CNS and inducing an immune-suppressive microenvironment in the SLOs would provide an effective therapy for MS. FTY720 (fingolimod) and anti-integrin α4 antibody (natalizumab) are used in the clinic for treating MS 4, 5 , sequestering lymphocytes in the LNs and preventing them from reacting with autoantigens in target tissues. Experimental autoimmune encephalomyelitis (EAE) is a widely accepted murine model of MS, reflecting many features of disease progression and developmental mechanism, including lymphocyte migration to the CNS and demyelination.Interleukin (IL)-4 is a pleiotropic anti-inflammatory cytokine that differentiates naïve CD4 + T cells into a Th2 phenotype and results in decreased differentiation into Th1 and Th17 6 . IL-4 suppresses re-activation of committed Th17 cells 6 . Moreover, IL-4 polarizes macrophages toward the M2 phenotype, an antiinflammatory phenotype 7 . IL-4 has been reported to suppress EAE disease incidence and severity 8,9 ; this occurs not only through direct immuno-modulation, as a recent study has shown that intranasally administered IL-4 improved disease outcomes of EAE through IL-4 directly binding to neurons to promote regeneration 10 .Although IL-4 stimulates multiple pathways to suppress EAE, it has yet to be tr...

Objective. Rheumatoid arthritis (RA) is a major autoimmune disease that causes synovitis and joint damage. Although clinical trials have been performed using interleukin-10 (IL-10), an antiinflammatory cytokine, as a potential treatment of RA, the therapeutic effects of IL-10 have been limited, potentially due to insufficient residence in lymphoid organs, where antigen recognition primarily occurs. This study was undertaken to engineer an IL-10-serum albumin (SA) fusion protein and evaluate its effects in 2 murine models of RA.Methods. SA-fused IL-10 (SA-IL-10) was recombinantly expressed. Mice with collagen antibody-induced arthritis (n = 4-7 per group) or collagen-induced arthritis (n = 9-15 per group) were injected intravenously with wild-type IL-10 or SA-IL-10, and the retention of SA-IL-10 in the lymph nodes (LNs), immune cell composition in the paws, and therapeutic effect of SA-IL-10 on mice with arthritis were assessed.Results. SA fusion to IL-10 led to enhanced accumulation in the mouse LNs compared with unmodified IL-10. Intravenous SA-IL-10 treatment restored immune cell composition in the paws to a normal status, elevated the frequency of suppressive alternatively activated macrophages, reduced IL-17A levels in the paw-draining LN, and protected joint morphology. Intravenous SA-IL-10 treatment showed similar efficacy as treatment with an anti-tumor necrosis factor antibody. SA-IL-10 was equally effective when administered intravenously, locally, or subcutaneously, which is a benefit for clinical translation of this molecule.Conclusion. SA fusion to IL-10 is a simple but effective engineering strategy for RA therapy and has potential for clinical translation.

The SARS-CoV-2 virus has caused an unprecedented global crisis, and curtailing its spread requires an effective vaccine which elicits a diverse and robust immune response. We have previously shown that vaccines made of a polymeric glyco-adjuvant conjugated to an antigen were effective in triggering such a response in other disease models and hypothesized that the technology could be adapted to create an effective vaccine against SARS-CoV-2. The core of the vaccine platform is the copolymer p(Man-TLR7), composed of monomers with pendant mannose or a toll-like receptor 7 (TLR7) agonist. Thus, p(Man-TLR7) is designed to target relevant antigen-presenting cells (APCs) via mannose-binding receptors and then activate TLR7 upon endocytosis. The p(Man-TLR7) construct is amenable to conjugation to protein antigens such as the Spike protein of SARS-CoV-2, yielding Spike-p(Man-TLR7). Here, we demonstrate Spike-p(Man-TLR7) vaccination elicits robust antigen-specific cellular and humoral responses in mice. In adult and elderly wild-type mice, vaccination with Spike-p(Man-TLR7) generates high and long-lasting titers of anti-Spike IgGs, with neutralizing titers exceeding levels in convalescent human serum. Interestingly, adsorbing Spike-p(Man-TLR7) to the depot-forming adjuvant alum amplified the broadly neutralizing humoral responses to levels matching those in mice vaccinated with formulations based off of clinically-approved adjuvants. Additionally, we observed an increase in germinal center B cells, antigen-specific antibody secreting cells, activated T follicular helper cells, and polyfunctional Th1-cytokine producing CD4 + and CD8 + T cells. We conclude that Spike-p(Man-TLR7) is an attractive, next-generation subunit vaccine candidate, capable of inducing durable and robust antibody and T cell responses.

The COVID-19 pandemic underscores the need for rapid, safe, and effective vaccines. In contrast to some traditional vaccines, nanoparticle-based subunit vaccines are particularly efficient in trafficking antigens to lymph nodes, where they induce potent immune cell activation. Here, we developed a strategy to decorate the surface of oxidation-sensitive polymersomes with multiple copies of the SARS-CoV-2 spike protein receptor-binding domain (RBD) to mimic the physical form of a virus particle. We evaluated the vaccination efficacy of these surface-decorated polymersomes (RBD surf ) in mice compared to RBD-encapsulated polymersomes (RBD encap ) and unformulated RBD (RBD free ), using monophosphoryl-lipid-A-encapsulated polymersomes (MPLA PS) as an adjuvant. While all three groups produced high titers of RBD-specific IgG, only RBD surf elicited a neutralizing antibody response to SARS-CoV-2 comparable to that of human convalescent plasma. Moreover, RBD surf was the only group to significantly increase the proportion of RBD-specific germinal center B cells in the vaccination-site draining lymph nodes. Both RBD surf and RBD encap drove similarly robust CD4 + and CD8 + T cell responses that produced multiple Th1-type cytokines. We conclude that a multivalent surface display of spike RBD on polymersomes promotes a potent neutralizing antibody response to SARS-CoV-2, while both antigen formulations promote robust T cell immunity.