Tregs integrate native and CAR-mediated costimulatory signals for control of allograft rejection.

Tregs expressing chimeric antigen receptors (CAR-Tregs) are a promising tool to promote transplant tolerance. The relationship between CAR structure and Treg function was studied in xenogeneic, immunodeficient mice, revealing advantages of CD28-encoding CARs. However, these models could underrepresent interactions between CAR-Tregs, antigen-presenting cells (APCs), and donor-specific Abs. We generated Tregs expressing HLA-A2-specific CARs with different costimulatory domains and compared their function in vitro and in vivo using an immunocompetent model of transplantation. In vitro, the CD28-encoding CAR had superior antigen-specific suppression, proliferation, and cytokine production. In contrast, in vivo, Tregs expressing CARs encoding CD28, ICOS, programmed cell death 1, and GITR, but not 4-1BB or OX40, all extended skin allograft survival. To reconcile in vitro and in vivo data, we analyzed effects of a CAR encoding CD3ζ but no costimulatory domain. These data revealed that exogenous costimulation from APCs can compensate for the lack of a CAR-encoded CD28 domain. Thus, Tregs expressing a CAR with or without CD28 are functionally equivalent in vivo, mediating similar extension of skin allograft survival and controlling the generation of anti-HLA-A2 alloantibodies. This study reveals a dimension of CAR-Treg biology and has important implications for the design of CARs for clinical use in Tregs.

Bioengineered particles expand myelin-specific regulatory T cells and reverse autoreactivity in a mouse model of multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune disease characterized by autoreactive immune cells damaging myelinated nerves, impairing brain function. Treatments aim for tolerance induction to reeducate the immune system to recognize myelin as "self" rather than "foreign." As peripheral immune tolerance is primarily mediated by regulatory T cells (Tregs), we developed a therapy to support Treg expansion and activity in vivo. To target, engage, and activate myelin-specific Tregs, we designed a biodegradable microparticle (MP) loaded with rapamycin and functionalized with a biased interleukin-2 (IL-2) fusion protein and a major histocompatibility complex (MHC) class II loaded with a myelin peptide. These tolerogenic MPs (Tol-MPs) were validated in vitro and then evaluated in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Tol-MPs promoted sustained disease reversal in 100% of mice and full recovery in 38% of mice with symptomatic EAE. Tol-MPs are a promising platform for treatment of autoimmune diseases.

Design and characterization of lipid nanocarriers for oral delivery of immunotherapeutic peptides.

The use of therapeutic proteins and peptides is of great interest for the treatment of many diseases, and advances in nanotechnology offer a path toward their stable delivery via preferred routes of administration. In this study, we sought to design and formulate a nanostructured lipid carrier (NLC) containing a nominal antigen (insulin peptide) for oral delivery. We utilized the design of experiments (DOE) statistical method to determine the dependencies of formulation variables on physicochemical particle characteristics including particle size, polydispersity (PDI), melting point, and latent heat of melting. The particles were determined to be non-toxic in vitro, readily taken up by primary immune cells, and found to accumulate in regional lymph nodes following oral administration. We believe that this platform technology could be broadly useful for the treatment of autoimmune diseases by supporting the development of oral delivery-based antigen specific immunotherapies.

Engineered human cytokine/antibody fusion proteins expand regulatory T cells and confer autoimmune disease protection.

Low-dose human interleukin-2 (hIL-2) treatment is used clinically to treat autoimmune disorders due to the cytokine's preferential expansion of immunosuppressive regulatory T cells (Tregs). However, off-target immune cell activation and short serum half-life limit the clinical potential of IL-2 treatment. Recent work showed that complexes comprising hIL-2 and the anti-hIL-2 antibody F5111 overcome these limitations by preferentially stimulating Tregs over immune effector cells. Although promising, therapeutic translation of this approach is complicated by the need to optimize dosing ratios and by the instability of the cytokine/antibody complex. We leverage structural insights to engineer a single-chain hIL-2/F5111 antibody fusion protein, termed F5111 immunocytokine (IC), which potently and selectively activates and expands Tregs. F5111 IC confers protection in mouse models of colitis and checkpoint inhibitor-induced diabetes mellitus. These results provide a roadmap for IC design and establish a Treg-biased immunotherapy that could be clinically translated for autoimmune disease treatment.

Targeting inflammation and immune activation to improve CTLA4-Ig-based modulation of transplant rejection.

For the last few decades, Calcineurin inhibitors (CNI)-based therapy has been the pillar of immunosuppression for prevention of organ transplant rejection. However, despite exerting effective control of acute rejection in the first year post-transplant, prolonged CNI use is associated with significant side effects and is not well suited for long term allograft survival. The implementation of Costimulation Blockade (CoB) therapies, based on the interruption of T cell costimulatory signals as strategy to control allo-responses, has proven potential for better management of transplant recipients compared to CNI-based therapies. The use of the biologic cytotoxic T-lymphocyte associated protein 4 (CTLA4)-Ig is the most successful approach to date in this arena. Following evaluation of the BENEFIT trials, Belatacept, a high-affinity version of CTLA4-Ig, has been FDA approved for use in kidney transplant recipients. Despite its benefits, the use of CTLA4-Ig as a monotherapy has proved to be insufficient to induce long-term allograft acceptance in several settings. Multiple studies have demonstrated that events that induce an acute inflammatory response with the consequent release of proinflammatory cytokines, and an abundance of allograft-reactive memory cells in the recipient, can prevent the induction of or break established immunomodulation induced with CoB regimens. This review highlights advances in our understanding of the factors and mechanisms that limit CoB regimens efficacy. We also discuss recent successes in experimentally designing complementary therapies that favor CTLA4-Ig effect, affording a better control of transplant rejection and supporting their clinical applicability.

Modeling the Potential of Treg-Based Therapies for Transplant Rejection: Effect of Dose, Timing, and Accumulation Site.

Introduction: The adoptive transfer of regulatory T cells (Tregs) has emerged as a method to promote graft tolerance. Clinical trials have demonstrated the safety of adoptive transfer and are now assessing their therapeutic efficacy. Strategies that generate large numbers of antigen specific Tregs are even more efficacious. However, the combinations of factors that influence the outcome of adoptive transfer are too numerous to be tested experimentally. Here, mathematical modeling is used to predict the most impactful treatment scenarios. Methods: We adapted our mathematical model of murine heart transplant rejection to simulate Treg adoptive transfer and to correlate therapeutic efficacy with Treg dose and timing, frequency of administration, and distribution of injected cells. Results: The model predicts that Tregs directly accumulating to the graft are more protective than Tregs localizing to draining lymph nodes. Inhibiting antigen-presenting cell maturation and effector functions at the graft site was more effective at modulating rejection than inhibition of T cell activation in lymphoid tissues. These complex dynamics define non-intuitive relationships between graft survival and timing and frequency of adoptive transfer. Conclusion: This work provides the framework for better understanding the impact of Treg adoptive transfer and will guide experimental design to improve interventions.

A short course of tofacitinib sustains the immunoregulatory effect of CTLA4-Ig in the presence of inflammatory cytokines and promotes long-term survival of murine cardiac allografts.

Costimulation blockade-based regimens are a promising strategy for management of transplant recipients. However, maintenance immunosuppression via CTLA4-Ig monotherapy is characterized by high frequency of rejection episodes. Recent evidence suggests that inflammatory cytokines contribute to alloreactive T cell activation in a CD28-independent manner, a reasonable contributor to the limited efficacy of CTLA4-Ig. In this study, we investigated the possible synergism of a combined short-term inhibition of cytokine signaling and CD28 engagement on the modulation of rejection. Our results demonstrate that the JAK/STAT inhibitor tofacitinib restored the immunomodulatory effect of CTLA4-Ig on mouse alloreactive T cells in the presence of inflammatory cytokines. Tofacitinib exposure conferred dendritic cells with a tolerogenic phenotype reducing their cytokine secretion and costimulatory molecules expression. JAK inhibition also directly affected T cell activation. In vivo, the combination of CTLA4-Ig and tofacitinib induced long-term survival of heart allografts and, importantly, it was equally effective when using grafts subjected to prolonged ischemia. Transplant survival correlated with a reduction in effector T cells and intragraft accumulation of regulatory T cells. Collectively, our studies demonstrate a powerful synergism between CTLA4-Ig and tofacitinib and suggest their combined use is a promising strategy for improved management of transplanted patients.

Jakinibs of All Trades: Inhibiting Cytokine Signaling in Immune-Mediated Pathologies.

Over the last 25 years, inhibition of Janus kinases (JAKs) has been pursued as a modality for treating various immune and inflammatory disorders. While the clinical development of JAK inhibitors (jakinibs) began with the investigation of their use in allogeneic transplantation, their widest successful application came in autoimmune and allergic diseases. Multiple molecules have now been approved for diseases ranging from rheumatoid and juvenile arthritis to ulcerative colitis, atopic dermatitis, graft-versus-host-disease (GVHD) and other inflammatory pathologies in 80 countries around the world. Moreover, two jakinibs have also shown surprising efficacy in the treatment of hospitalized coronavirus disease-19 (COVID-19) patients, indicating additional roles for jakinibs in infectious diseases, cytokine storms and other hyperinflammatory syndromes. Jakinibs, as a class of pharmaceutics, continue to expand in clinical applications and with the development of more selective JAK-targeting and organ-selective delivery. Importantly, jakinib safety and pharmacokinetics have been investigated alongside clinical development, further cementing the potential benefits and limits of jakinib use. This review covers jakinibs that are approved or are under late phase investigation, focusing on clinical applications, pharmacokinetic and safety profiles, and future opportunities and challenges.

Multiphase Assembly of Small Molecule Microcrystalline Peptide Hydrogel Allows Immunomodulatory Combination Therapy for Long-Term Heart Transplant Survival.

Combination therapies that target multiple pathways involved in immune rejection of transplants hold promise for patients in need of restorative surgery. Herein, a noninteracting multiphase molecular assembly approach is developed to crystallize tofacitinib, a potent JAK1/3 inhibitor, within a shear-thinning self-assembled fibrillar peptide hydrogel network. The resulting microcrystalline tofacitinib hydrogel (MTH) can be syringe-injected directly to the grafting site during surgery to locally deliver the small molecule. The rate of drug delivered from MTH is largely controlled by the dissolution of the encapsulated microcrystals. A single application of MTH, in combination with systemically delivered CTLA4-Ig, a co-stimulation inhibitor, affords significant graft survival in mice receiving heterotopic heart transplants. Locoregional studies indicate that the local delivery of tofacitinib at the graft site enabled by MTH is required for the observed enhanced graft survival.

Vascularized composite allotransplantation combined with costimulation blockade induces mixed chimerism and reveals intrinsic tolerogenic potential.

Vascularized composite allotransplantation (VCA) has become a valid therapeutic option to restore form and function after devastating tissue loss. However, the need for high-dose multidrug immunosuppression to maintain allograft survival is still hampering more widespread application of VCA. In this study, we investigated the immunoregulatory potential of costimulation blockade (CoB; CTLA4-Ig and anti-CD154 mAb) combined with nonmyeoablative total body irradiation (TBI) to promote allograft survival of VCA in a fully MHC-mismatched mouse model of orthotopic hind limb transplantation. Compared with untreated controls (median survival time [MST] 8 days) and CTLA4-Ig treatment alone (MST 17 days), CoB treatment increased graft survival (MST 82 days), and the addition of nonmyeloablative TBI led to indefinite graft survival (MST > 210 days). Our analysis suggests that VCA-derived BM induced mixed chimerism in animals treated with CoB and TBI + CoB, promoting gradual deletion of alloreactive T cells as the underlying mechanism of long-term allograft survival. Acceptance of donor-matched secondary skin grafts, decreased ex vivo T cell responsiveness, and increased graft-infiltrating Tregs further indicated donor-specific tolerance induced by TBI + CoB. In summary, our data suggest that vascularized BM-containing VCAs are immunologically favorable grafts promoting chimerism induction and long-term allograft survival in the context of CoB.

A computational model for microcirculation including Fahraeus-Lindqvist effect, plasma skimming and fluid exchange with the tissue interstitium.

We present a two-phase model for microcirculation that describes the interaction of plasma with red blood cells. The model takes into account of typical effects characterizing the microcirculation, such as the Fahraeus-Lindqvist effect and plasma skimming. Besides these features, the model describes the interaction of capillaries with the surrounding tissue. More precisely, the model accounts for the interaction of capillary transmural flow with the surrounding interstitial pressure. Furthermore, the capillaries are represented as one-dimensional channels with arbitrary, possibly curved configuration. The latter two features rely on the unique ability of the model to account for variations of flow rate and pressure along the axis of the capillary, according to a local differential formulation of mass and momentum conservation. Indeed, the model stands on a solid mathematical foundation, which is also addressed in this work. In particular, we present the model derivation, the variational formulation, and its approximation using the finite element method. Finally, we conclude the work with a comparative computational study of the importance of the Fahraeus-Lindqvist, plasma skimming, and capillary leakage effects on the distribution of flow in a microvascular network.

Type-I Interferons Inhibit Interleukin-10 Signaling and Favor Type 1 Diabetes Development in Nonobese Diabetic Mice.

Destruction of insulin-producing ß-cells by autoreactive T lymphocytes leads to the development of type 1 diabetes. Type-I interferons (TI-IFN) and interleukin-10 (IL-10) have been connected with the pathophysiology of this disease; however, their interplay in the modulation of diabetogenic T cells remains unknown. We have discovered that TI-IFN cause a selective inhibition of IL-10 signaling in effector and regulatory T cells, altering their responses. This correlates with diabetes development in nonobese diabetic mice, where the inhibition is also spatially localized to T cells of pancreatic and mesenteric lymph nodes. IL-10 signaling inhibition is reversible and can be restored via blockade of TI-IFN/IFN-R interaction, paralleling with the resulting delay in diabetes onset and reduced severity. Overall, we propose a novel molecular link between TI-IFN and IL-10 signaling that helps better understand the complex dynamics of autoimmune diabetes development and reveals new strategies of intervention.

Desensitization and Prevention of Antibody-Mediated Rejection in Vascularized Composite Allotransplantation by Syngeneic Hematopoietic Stem Cell Transplantation.

BACKGROUND: Candidates for vascularized composite allotransplantation (VCA) are frequently sensitized, putting them at risk for antibody-mediated rejection. Current desensitization strategies are imperfect and require a living-donor setting. Here we investigated the impact of sensitization on and the efficacy of a desensitization protocol utilizing syngeneic hematopoietic stem cell transplantation (HSCT) to prevent antibody-mediated rejection in VCA. METHODS: Skin transplants from Dark Agouti to Lewis rats were performed for sensitization. Orthotopic hind limb transplants from Dark Agouti donors were performed to sensitized and nonsensitized recipients, and the animals were treated with either daily tacrolimus or no immunosuppression. A desensitization protocol consisting of total body irradiation, fludarabine, and syngeneic HSCT was applied to sensitized animals. Graft rejection was monitored by clinical assessment and histological analysis. Serum levels of donor-specific antibodies (DSA IgG) were measured using flow cytometry. RESULTS: Sensitized recipients exhibited accelerated rejection by 5.5 ± 1.2 days without immunosuppression and 10.2 ± 3.6 days with daily tacrolimus compared with 8.7 ± 1.2 days and longer than 30 days in nonsensitized recipients, respectively. Serum levels of DSA IgG were markedly elevated (37.3 ± 3.34-fold from baseline) in sensitized recipients after VCA and correlated with histologic evidence of rejection and C4d deposition. Desensitization significantly reduced DSA compared with sensitized controls (2.6 ± 0.5-fold vs 6.0 ± 1.2-fold, P < 0.01) and along with daily tacrolimus led to improved VCA survival longer than 30 days without evidence of C4d deposition (n = 6). CONCLUSIONS: In summary, sensitization leads to accelerated rejection of VCA, and syngeneic HSCT combined with conventional immunosuppression effectively reduces DSA and improves allograft survival in sensitized rats.

Mechanisms of rejection in vascular composite allotransplantation.

PURPOSE OF REVIEW: For patients with devastating injuries in whom standard reconstruction is not an option, vascularized composite allotransplantation (VCA) has become a viable means of restoring form and function. However, immunological rejection continues to be a problem in VCA and has not yet been fully characterized. As the field is relatively new, much of the data on rejection and immunosuppression have been extrapolated from that of solid organ transplantation. In this review, we cover the basic mechanisms of rejection as they relate specifically to VCA with analysis of recent literature and future directions. RECENT FINDINGS: Recent clinical studies have supported previously postulated T-cell-mediated mechanism of acute rejection and have also made strides in differentiating rejection from inflammation from other skin conditions and with different treatment regimens. Antibody-mediated rejection has been described in recent cases as well as treatment of presensitized patients receiving VCAs. With more long-term grafts, chronic changes, including vasculopathy, are being reported. SUMMARY: Clinically observed types of rejection in VCA include mainly cell-mediated, antibody-mediated and chronic rejection. Advances in diagnosis and treatment of rejection have been made, but there is still much to be learned about VCA-specific rejection.

Combining Theoretical and Experimental Techniques to Study Murine Heart Transplant Rejection.

The quality of life of organ transplant recipients is compromised by complications associated with life-long immunosuppression, such as hypertension, diabetes, opportunistic infections, and cancer. Moreover, the absence of established tolerance to the transplanted tissues causes limited long-term graft survival rates. Thus, there is a great medical need to understand the complex immune system interactions that lead to transplant rejection so that novel and effective strategies of intervention that redirect the system toward transplant acceptance (while preserving overall immune competence) can be identified. This study implements a systems biology approach in which an experimentally based mathematical model is used to predict how alterations in the immune response influence the rejection of mouse heart transplants. Five stages of conventional mouse heart transplantation are modeled using a system of 13 ordinary differential equations that tracks populations of both innate and adaptive immunity as well as proxies for pro- and anti-inflammatory factors within the graft and a representative draining lymph node. The model correctly reproduces known experimental outcomes, such as indefinite survival of the graft in the absence of CD4+ T cells and quick rejection in the absence of CD8+ T cells. The model predicts that decreasing the translocation rate of effector cells from the lymph node to the graft delays transplant rejection. Increasing the starting number of quiescent regulatory T cells in the model yields a significant but somewhat limited protective effect on graft survival. Surprisingly, the model shows that a delayed appearance of alloreactive T cells has an impact on graft survival that does not correlate linearly with the time delay. This computational model represents one of the first comprehensive approaches toward simulating the many interacting components of the immune system. Despite some limitations, the model provides important suggestions of experimental investigations that could improve the understanding of rejection. Overall, the systems biology approach used here is a first step in predicting treatments and interventions that can induce transplant tolerance while preserving the capacity of the immune system to protect against legitimate pathogens.

A Novel mTORC1-Dependent, Akt-Independent Pathway Differentiates the Gut Tropism of Regulatory and Conventional CD4 T Cells.

The vitamin A metabolite all-trans retinoic acid (ATRA) induces a gut-homing phenotype in activated CD4(+) conventional T cells (Tconv) by upregulating the integrin α4ß7 and the chemokine receptor CCR9. We report that, in contrast to mouse Tconv, only ∼50% of regulatory T cells (Treg) upregulate CCR9 when stimulated by physiological levels of ATRA, even though Tconv and Treg express similar levels of the retinoic acid receptor (RAR). The resulting bimodal CCR9 expression is not associated with differences in the extent of their proliferation, level of Foxp3 expression, or affiliation with naturally occurring Treg or induced Treg in the circulating Treg pool. Furthermore, we find that exposure of Treg to the mechanistic target of rapamycin (mTOR) inhibitor rapamycin suppresses upregulation of both CCR9 and α4ß7, an effect that is not evident with Tconv. This suggests that in Treg, ATRA-induced upregulation of CCR9 and α4ß7 is dependent on activation of a mTOR signaling pathway. The involvement of mTOR is independent of Akt activity, because specific inhibition of Akt, pyruvate dehydrogenase kinase-1, or its downstream target glycogen synthase kinase-3 did not prevent CCR9 expression. Additionally, Rictor (mTOR complex [mTORC]2)-deficient Treg showed unaltered ability to express CCR9, whereas Raptor (mTORC1)-deficient Treg were unable to upregulate CCR9, suggesting the selective participation of mTORC1. These findings reveal a novel difference between ATRA signaling and chemokine receptor induction in Treg versus Tconv and provide a framework via which the migratory behavior of Treg versus Tconv might be regulated differentially for therapeutic purposes.

Orthotopic Hind Limb Transplantation in the Mouse.

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications.

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.