Early Immunomodulatory Program Triggered by Protolerogenic Bifidobacterium pseudolongum Drives Cardiac Transplant Outcomes.

BACKGROUND: Despite ongoing improvements to regimens preventing allograft rejection, most cardiac and other organ grafts eventually succumb to chronic vasculopathy, interstitial fibrosis, or endothelial changes, and eventually graft failure. The events leading to chronic rejection are still poorly understood and the gut microbiota is a known driving force in immune dysfunction. We previously showed that gut microbiota dysbiosis profoundly influences the outcome of vascularized cardiac allografts and subsequently identified biomarker species associated with these differential graft outcomes. METHODS: In this study, we further detailed the multifaceted immunomodulatory properties of protolerogenic and proinflammatory bacterial species over time, using our clinically relevant model of allogenic heart transplantation. RESULTS: In addition to tracing longitudinal changes in the recipient gut microbiome over time, we observed that Bifidobacterium pseudolongum induced an early anti-inflammatory phenotype within 7 d, whereas Desulfovibrio desulfuricans resulted in a proinflammatory phenotype, defined by alterations in leukocyte distribution and lymph node (LN) structure. Indeed, in vitro results showed that B pseudolongum and D desulfuricans acted directly on primary innate immune cells. However, by 40 d after treatment, these 2 bacterial strains were associated with mixed effects in their impact on LN architecture and immune cell composition and loss of colonization within gut microbiota, despite protection of allografts from inflammation with B pseudolongum treatment. CONCLUSIONS: These dynamic effects suggest a critical role for early microbiota-triggered immunologic events such as innate immune cell engagement, T-cell differentiation, and LN architectural changes in the subsequent modulation of protolerant versus proinflammatory immune responses in organ transplant recipients.

Rapid intestinal and systemic metabolic reprogramming in an immunosuppressed environment.

Intrinsic metabolism shapes the immune environment associated with immune suppression and tolerance in settings such as organ transplantation and cancer. However, little is known about the metabolic activities in an immunosuppressive environment. In this study, we employed metagenomic, metabolomic, and immunological approaches to profile the early effects of the immunosuppressant drug tacrolimus, antibiotics, or both in gut lumen and circulation using a murine model. Tacrolimus induced rapid and profound alterations in metabolic activities within two days of treatment, prior to alterations in gut microbiota composition and structure. The metabolic profile and gut microbiome after seven days of treatment was distinct from that after two days of treatment, indicating continuous drug effects on both gut microbial ecosystem and host metabolism. The most affected taxonomic groups are Clostriales and Verrucomicrobiae (i.e., Akkermansia muciniphila), and the most affected metabolic pathways included a group of interconnected amino acids, bile acid conjugation, glucose homeostasis, and energy production. Highly correlated metabolic changes were observed between lumen and serum metabolism, supporting their significant interactions. Despite a small sample size, this study explored the largely uncharacterized microbial and metabolic events in an immunosuppressed environment and demonstrated that early changes in metabolic activities can have significant implications that may serve as antecedent biomarkers of immune activation or quiescence. To understand the intricate relationships among gut microbiome, metabolic activities, and immune cells in an immune suppressed environment is a prerequisite for developing strategies to monitor and optimize alloimmune responses that determine transplant outcomes.

Rapid intestinal and systemic metabolic reprogramming in an immunosuppressed environment.

Intrinsic metabolism shapes the immune environment associated with immune suppression and tolerance in settings such as organ transplantation and cancer. However, little is known about the metabolic activities in an immunosuppressive environment. In this study, we employed metagenomic, metabolomic, and immunological approaches to profile the early effects of the immunosuppressant drug tacrolimus, antibiotics, or both in gut lumen and circulation using a murine model. Tacrolimus induced rapid and profound alterations in metabolic activities within two days of treatment, prior to alterations in gut microbiota composition and structure. The metabolic profile and gut microbiome after seven days of treatment was distinct from that after two days of treatment, indicating continuous drug effects on both gut microbial ecosystem and host metabolism. The most affected taxonomic groups are Clostriales and Verrucomicrobiae (i.e., Akkermansia muciniphila), and the most affected metabolic pathways included a group of interconnected amino acids, bile acid conjugation, glucose homeostasis, and energy production. Highly correlated metabolic changes were observed between lumen and serum metabolism, supporting their significant interactions. Despite a small sample size, this study explored the largely uncharacterized microbial and metabolic events in an immunosuppressed environment and demonstrated that early changes in metabolic activities can have significant implications that may serve as antecedent biomarkers of immune activation or quiescence. To understand the intricate relationships among gut microbiome, metabolic activities, and immune cells in an immune suppressed environment is a prerequisite for developing strategies to monitor and optimize alloimmune responses that determine transplant outcomes.

Engineering the lymph node environment promotes antigen-specific efficacy in type 1 diabetes and islet transplantation.

Antigen-specific tolerance is a key goal of experimental immunotherapies for autoimmune disease and allograft rejection. This outcome could selectively inhibit detrimental inflammatory immune responses without compromising functional protective immunity. A major challenge facing antigen-specific immunotherapies is ineffective control over immune signal targeting and integration, limiting efficacy and causing systemic non-specific suppression. Here we use intra-lymph node injection of diffusion-limited degradable microparticles that encapsulate self-antigens with the immunomodulatory small molecule, rapamycin. We show this strategy potently inhibits disease during pre-clinical type 1 diabetes and allogenic islet transplantation. Antigen and rapamycin are required for maximal efficacy, and tolerance is accompanied by expansion of antigen-specific regulatory T cells in treated and untreated lymph nodes. The antigen-specific tolerance in type 1 diabetes is systemic but avoids non-specific immune suppression. Further, microparticle treatment results in the development of tolerogenic structural microdomains in lymph nodes. Finally, these local structural and functional changes in lymph nodes promote memory markers among antigen-specific regulatory T cells, and tolerance that is durable. This work supports intra-lymph node injection of tolerogenic microparticles as a powerful platform to promote antigen-dependent efficacy in type 1 diabetes and allogenic islet transplantation.

Strain-specific alterations in gut microbiome and host immune responses elicited by tolerogenic Bifidobacterium pseudolongum.

The beneficial effects attributed to Bifidobacterium are largely attributed to their immunomodulatory capabilities, which are likely to be species- and even strain-specific. However, their strain-specificity in direct and indirect immune modulation remain largely uncharacterized. We have shown that B. pseudolongum UMB-MBP-01, a murine isolate strain, is capable of suppressing inflammation and reducing fibrosis in vivo. To ascertain the mechanism driving this activity and to determine if it is specific to UMB-MBP-01, we compared it to a porcine tropic strain B. pseudolongum ATCC25526 using a combination of cell culture and in vivo experimentation and comparative genomics approaches. Despite many shared features, we demonstrate that these two strains possess distinct genetic repertoires in carbohydrate assimilation, differential activation signatures and cytokine responses signatures in innate immune cells, and differential effects on lymph node morphology with unique local and systemic leukocyte distribution. Importantly, the administration of each B. pseudolongum strain resulted in major divergence in the structure, composition, and function of gut microbiota. This was accompanied by markedly different changes in intestinal transcriptional activities, suggesting strain-specific modulation of the endogenous gut microbiota as a key to immune modulatory host responses. Our study demonstrated a single probiotic strain can influence local, regional, and systemic immunity through both innate and adaptive pathways in a strain-specific manner. It highlights the importance to investigate both the endogenous gut microbiome and the intestinal responses in response to probiotic supplementation, which underpins the mechanisms through which the probiotic strains drive the strain-specific effect to impact health outcomes.

Treg tissue stability depends on lymphotoxin beta-receptor- and adenosine-receptor-driven lymphatic endothelial cell responses.

Regulatory T cell (Treg) lymphatic migration is required for resolving inflammation and prolonging allograft survival. Focusing on Treg interactions with lymphatic endothelial cells (LECs), we dissect mechanisms and functional consequences of Treg transendothelial migration (TEM). Using three genetic mouse models of pancreatic islet transplantation, we show that Treg lymphotoxin (LT) αß and LEC LTß receptor (LTßR) signaling are required for efficient Treg migration and suppressive function to prolong allograft survival. Inhibition of LT signaling increases Treg conversion to Foxp3loCD25lo exTregs. In a transwell-based model of TEM across polarized LECs, non-migrated Tregs become exTregs. Such conversion is regulated by LTßR nuclear factor κB (NF-κB) signaling in LECs, which increases interleukin-6 (IL-6) production and drives exTreg conversion. Migrating Tregs are ectonucleotidase CD39hi and resist exTreg conversion in an adenosine-receptor-2A-dependent fashion. Human Tregs migrating across human LECs behave similarly. These molecular interactions can be targeted for therapeutic manipulation of immunity and suppression.

PD-L1 signaling selectively regulates T cell lymphatic transendothelial migration.

Programmed death-1 (PD-1) and its ligand PD-L1 are checkpoint molecules which regulate immune responses. Little is known about their functions in T cell migration and there are contradictory data about their roles in regulatory T cell (Treg) function. Here we show activated Tregs and CD4 effector T cells (Teffs) use PD-1/PD-L1 and CD80/PD-L1, respectively, to regulate transendothelial migration across lymphatic endothelial cells (LECs). Antibody blockade of Treg PD-1, Teff CD80 (the alternative ligand for PD-L1), or LEC PD-L1 impairs Treg or Teff migration in vitro and in vivo. PD-1/PD-L1 signals through PI3K/Akt and ERK to regulate zipper junctional VE-cadherin, and through NFκB-p65 to up-regulate VCAM-1 expression on LECs. CD80/PD-L1 signaling up-regulates VCAM-1 through ERK and NFκB-p65. PD-1 and CD80 blockade reduces tumor egress of PD-1high fragile Tregs and Teffs into draining lymph nodes, respectively, and promotes tumor regression. These data provide roles for PD-L1 in cell migration and immune regulation.

Low-Risk Papillary Thyroid Cancer: Treatment De-Escalation and Cost Implications.

INTRODUCTION: The American Thyroid Association (ATA) updated consensus guidelines in 2015 for radioactive iodine (RAI) and resection for low-risk papillary thyroid cancer. The objective of this study was to describe the evolution of institutional practice patterns and estimate the cost implications of these trends. MATERIALS AND METHODS: Patients with cT1-T2N0 papillary thyroid cancer were identified via an institutional tumor registry. Incidences of total thyroidectomy or RAI were tracked longitudinally using cumulative sum. Real-world costs for RAI and each surgical encounter were adjusted for inflation and standardized to national average costs from National Inpatient Sample cost data. RESULTS: Sixty-one patients met inclusion criteria between 2007 and 2018. Among these, 28 patients underwent total thyroidectomies and received RAI treatments based on criteria pre-dating the 2015 ATA guidelines. Cumulative sum revealed significant decreases in the rate of total thyroidectomy following May 2015 (15.8% versus 59.5%, P = 0.002) and RAI following March 2013 (3.0% versus 32.1%, P = 0.002). There were no locoregional recurrences in either period. The average cost savings attributable to these institutional practice changes was $1580 per patient. CONCLUSIONS: De-escalation in surgical and RAI utilization for low-risk papillary thyroid cancer according to 2015 ATA guidelines is associated with a substantial decrease in real-world costs.

The lymph node stromal laminin α5 shapes alloimmunity.

Lymph node stromal cells (LNSCs) regulate immunity through constructing lymphocyte niches. LNSC-produced laminin α5 (Lama5) regulates CD4+ T cells but the underlying mechanisms of its functions are poorly understood. Here we show that depleting Lama5 in LNSCs resulted in decreased Lama5 protein in the LN cortical ridge (CR) and around high endothelial venules (HEVs). Lama5 depletion affected LN structure with increased HEVs, upregulated chemokines, and cell adhesion molecules, and led to greater numbers of Tregs in the T cell zone. Mouse and human T cell transendothelial migration and T cell entry into LNs were suppressed by Lama5 through the receptors α6 integrin and α-dystroglycan. During immune responses and allograft transplantation, depleting Lama5 promoted antigen-specific CD4+ T cell entry into the CR through HEVs, suppressed T cell activation, and altered T cell differentiation to suppressive regulatory phenotypes. Enhanced allograft acceptance resulted from depleting Lama5 or blockade of T cell Lama5 receptors. Lama5 and Lama4/Lama5 ratios in allografts were associated with the rejection severity. Overall, our results demonstrated that stromal Lama5 regulated immune responses through altering LN structures and T cell behaviors. This study delineated a stromal Lama5-T cell receptor axis that can be targeted for immune tolerance modulation.

Regulatory T Cells Condition Lymphatic Endothelia for Enhanced Transendothelial Migration.

Regulatory T cells (Tregs) express high levels of cell surface lymphotoxin alpha beta (LTα1ß2) to activate the LT beta receptor (LTßR) on the lymphatic endothelial cells (LECs), modulating LEC adhesion molecules, intercellular junctions, and chemokines. We demonstrate a role for Tregs through this pathway to condition the permissiveness of lymphatic endothelia for transendothelial migration (TEM), thus gating leukocyte traffic. Human Tregs share the same property with murine Tregs. Activation of TLR2 on Tregs during inflammation specifically augments LTα1ß2-LTßR signaling, which further enhances the permissiveness of LECs to facilitate TEM. The conditioning of endothelia may promote the resolution of inflammation by directing leukocytes out of tissues to lymphatic vessels and draining lymph nodes (dLNs). Thus, Tregs interact with lymphatic endothelia under homeostasis and inflammation and dictate endothelial permissiveness and gating mechanisms for subsequent leukocyte migration through endothelial barriers.

T Regulatory Cells and Priming the Suppressive Tumor Microenvironment.

Treg play a central role in maintenance of self tolerance and homeostasis through suppression of self-reactive T cell populations. In addition to that role, Treg also survey cancers and suppress anti-tumor immune responses. Thus, understanding the unique attributes of Treg-tumor interactions may permit control of this pathologic suppression without interfering with homeostatic self-tolerance. This review will define the unique role of Treg in cancer growth, and the ways by which Treg inhibit a robust anti-tumor immune response. There will be specific focus placed on Treg homing to the tumor microenvironment (TME), TME formation of induced Treg (iTreg), mechanisms of suppression that underpin cancer immune escape, and trophic nonimmunologic effects of Treg on tumor cells.

Role of lymph node stroma and microenvironment in T cell tolerance.

Lymph nodes (LNs) are at the cross roads of immunity and tolerance. These tissues are compartmentalized into specialized niche areas by lymph node stromal cells (LN SCs). LN SCs shape the LN microenvironment and guide immunological cells into different zones through establishment of a CCL19 and CCL21 gradient. Following local immunological cues, LN SCs modulate activity to support immune cell priming, activation, and fate. This review will present our current understanding of LN SC subsets roles in regulating T cell tolerance. Three major types of LN SC subsets, namely fibroblastic reticular cells, lymphatic endothelial cells, and blood endothelial cells, are discussed. These subsets serve as scaffolds to support and regulate T cell homeostasis. They contribute to tolerance by presenting peripheral tissue antigens to both CD4 and CD8 T cells. The role of LN SCs in regulating T cell migration and tolerance induction is discussed. Looking forward, recent advances in bioengineered materials and approaches to leverage LN SCs to induce T cell tolerance are highlighted, as are current clinical practices that allow for manipulation of the LN microenvironment to induce tolerance. Increased understanding of LN architecture, how different LN SCs integrate immunological cues and shape immune responses, and approaches to induce T cell tolerance will help further combat autoimmune diseases and graft rejection.