Human neutrophil peptides 1-3 protect the murine urinary tract from uropathogenic Escherichia coli challenge.

Antimicrobial peptides (AMPs) are critical to the protection of the urinary tract of humans and other animals from pathogenic microbial invasion. AMPs rapidly destroy pathogens by disrupting microbial membranes and/or augmenting or inhibiting the host immune system through a variety of signaling pathways. We have previously demonstrated that alpha-defensins 1-3 (DEFA1A3) are AMPs expressed in the epithelial cells of the human kidney collecting duct in response to uropathogens. We also demonstrated that DNA copy number variations in the DEFA1A3 locus are associated with UTI and pyelonephritis risk. Because DEFA1A3 is not expressed in mice, we utilized human DEFA1A3 gene transgenic mice (DEFA4/4) to further elucidate the biological relevance of this locus in the murine urinary tract. We demonstrate that the kidney transcriptional and translational expression pattern is similar in humans and the human gene transgenic mouse upon uropathogenic Escherichia coli (UPEC) stimulus in vitro and in vivo. We also demonstrate transgenic human DEFA4/4 gene mice are protected from UTI and pyelonephritis under various UPEC challenges. This study serves as the foundation to start the exploration of manipulating the DEFA1A3 locus and alpha-defensins 1-3 expression as a potential therapeutic target for UTIs and other infectious diseases.

Spatial multiomics of arterial regions from cardiac allograft vasculopathy rejected grafts reveal novel insights into the pathogenesis of chronic antibody-mediated rejection.

Cardiac allograft vasculopathy (CAV) causes late graft failure and mortality after heart transplantation. Donor-specific antibodies (DSAs) lead to chronic endothelial cell injury, inflammation, and arterial intimal thickening. In this study, GeoMx digital spatial profiling was used to analyze arterial areas of interest (AOIs) from CAV+DSA+ rejected cardiac allografts (N = 3; 22 AOIs total). AOIs were categorized based on CAV neointimal thickening and underwent whole transcriptome and protein profiling. By comparing our transcriptomic data with that of healthy control vessels of rapid autopsy myocardial tissue, we pinpointed specific pathways and transcripts indicative of heightened inflammatory profiles in CAV lesions. Moreover, we identified protein and transcriptomic signatures distinguishing CAV lesions exhibiting low and high neointimal lesions. AOIs with low neointima showed increased markers for activated inflammatory infiltrates, endothelial cell activation transcripts, and gene modules involved in metalloproteinase activation and TP53 regulation of caspases. Inflammatory and apoptotic proteins correlated with inflammatory modules in low neointima AOIs. High neointima AOIs exhibited elevated TGFß-regulated transcripts and modules enriched for platelet activation/aggregation. Proteins associated with growth factors/survival correlated with modules enriched for proliferation/repair in high neointima AOIs. Our findings reveal novel insight into immunological mechanisms mediating CAV pathogenesis.

Human leukocyte antigen class I antibody-activated endothelium promotes CD206+ M2 macrophage polarization and MMP9 secretion through TLR4 signaling and P-selectin in a model of antibody-mediated rejection and allograft vasculopathy.

HLA donor-specific antibodies (DSA) elicit alloimmune responses against the graft vasculature, leading to endothelial cell (EC) activation and monocyte infiltration during antibody-mediated rejection (AMR). AMR promotes chronic inflammation and remodeling, leading to thickening of the arterial intima termed transplant vasculopathy or cardiac allograft vasculopathy (CAV) in heart transplants. Intragraft-recipient macrophages serve as a diagnostic marker in AMR; however, their polarization and function remain unclear. In this study, we utilized an in vitro Transwell coculture system to explore the mechanisms of monocyte-to-macrophage polarization induced by HLA I DSA-activated ECs. Anti-HLA I (IgG or F(ab')2) antibody-activated ECs induced the polarization of M2 macrophages with increased CD206 expression and MMP9 secretion. However, inhibition of TLR4 signaling or PSGL-1-P-selectin interactions significantly decreased both CD206 and MMP9. Monocyte adherence to Fc-P-selectin coated plates induced M2 macrophages with increased CD206 and MMP9. Moreover, Fc-receptor and IgG interactions synergistically enhanced active-MMP9 in conjunction with P-selectin. Transcriptomic analysis of arteries from DSA+CAV+ rejected cardiac allografts and multiplex-immunofluorescent staining illustrated the expression of CD68+CD206+CD163+MMP9+ M2 macrophages within the neointima of CAV-affected lesions. These findings reveal a novel mechanism linking HLA I antibody-activated endothelium to the generation of M2 macrophages which secrete vascular remodeling proteins contributing to AMR and CAV pathogenesis.

Impact of Back-to-Base Normothermic Machine Perfusion on Complications and Costs: A Multicenter, Real-World Risk-Matched Analysis.

OBJECTIVE: Assess cost and complication outcomes after liver transplantation (LT) using normothermic machine perfusion (NMP). BACKGROUND: End-ischemic NMP is often used to aid logistics, yet its impact on outcomes after LT remains unclear, as does its true impact on costs associated with transplantation. METHODS: Deceased donor liver recipients at 2 centers (January 1, 2019, to June 30, 2023) were included. Retransplants, splits, and combined grafts were excluded. End-ischemic NMP (OrganOx-Metra) was implemented in October 2022 for extended-criteria donation after brain death (DBDs), all donations after circulatory deaths (DCDs), and logistics. NMP cases were matched 1:2 with static cold storage controls (SCS) using the Balance-of-Risk [donation after brain death (DBD)-grafts] and UK-DCD Score (DCD-grafts). RESULTS: Overall, 803 transplantations were included, 174 (21.7%) receiving NMP. Matching was achieved between 118 NMP-DBDs with 236 SCS; and 37 NMP-DCD with 74 corresponding SCS. For both graft types, median inpatient comprehensive complications index values were comparable between groups. DCD-NMP grafts experienced reduced cumulative 90-day comprehensive complications index (27.6 vs 41.9, P =0.028). NMP also reduced the need for early relaparotomy and renal replacement therapy, with subsequently less frequent major complications (Clavien-Dindo ≥IVa). This effect was more pronounced in DCD transplants. NMP had no protective effect on early biliary complications. Organ acquisition/preservation costs were higher with NMP, yet NMP-treated grafts had lower 90-day pretransplant costs in the context of shorter waiting list times. Overall costs were comparable for both cohorts. CONCLUSIONS: This is the first risk-adjusted outcome and cost analysis comparing NMP and SCS. In addition to logistical benefits, NMP was associated with a reduction in relaparotomy and bleeding in DBD grafts, and overall complications and post-LT renal replacement for DCDs. While organ acquisition/preservation was more costly with NMP, overall 90-day health care costs-per-transplantation were comparable.

Acute rejection after liver transplantation with machine perfusion versus static cold storage: A systematic review and meta-analysis.

BACKGROUND AND AIMS: Acute cellular rejection (ACR) is a frequent complication after liver transplantation. By reducing ischemia and graft damage, dynamic preservation techniques may diminish ACR. We performed a systematic review to assess the effect of currently tested organ perfusion (OP) approaches versus static cold storage (SCS) on post-transplant ACR-rates. APPROACH AND RESULTS: A systematic search of Medline, Embase, Cochrane Library, and Web of Science was conducted. Studies reporting ACR-rates between OP and SCS and comprising at least 10 liver transplants performed with either hypothermic oxygenated perfusion (HOPE), normothermic machine perfusion, or normothermic regional perfusion were included. Studies with mixed perfusion approaches were excluded. Eight studies were identified (226 patients in OP and 330 in SCS). Six studies were on HOPE, one on normothermic machine perfusion, and one on normothermic regional perfusion. At meta-analysis, OP was associated with a reduction in ACR compared with SCS [OR: 0.55 (95% CI, 0.33-0.91), p =0.02]. This effect remained significant when considering HOPE alone [OR: 0.54 (95% CI, 0.29-1), p =0.05], in a subgroup analysis of studies including only grafts from donation after cardiac death [OR: 0.43 (0.20-0.91) p =0.03], and in HOPE studies with only donation after cardiac death grafts [OR: 0.37 (0.14-1), p =0.05]. CONCLUSIONS: Dynamic OP techniques are associated with a reduction in ACR after liver transplantation compared with SCS. PROSPERO registration: CRD42022348356.

Infliximab Induction Lacks Efficacy and Increases BK Virus Infection in Deceased Donor Kidney Transplant Recipients: Results of the CTOT-19 Trial.

BACKGROUND: Ischemia-reperfusion (IR) of a kidney transplant (KTx) upregulates TNF α production that amplifies allograft inflammation and may negatively affect transplant outcomes. METHODS: We tested the effects of blocking TNF peri-KTx via a randomized, double-blind, placebo-controlled, 15-center, phase 2 clinical trial. A total of 225 primary transplant recipients of deceased-donor kidneys (KTx; 38.2% Black/African American, 44% White) were randomized to receive intravenous infliximab (IFX) 3 mg/kg or saline placebo (PLBO) initiated before kidney reperfusion. All patients received rabbit anti-thymocyte globulin induction and maintenance immunosuppression (IS) with tacrolimus, mycophenolate mofetil, and prednisone. The primary end point was the difference between groups in mean 24-month eGFR. RESULTS: There was no difference in the primary end point of 24-month eGFR between IFX (52.45 ml/min per 1.73 m 2 ; 95% CI, 48.38 to 56.52) versus PLBO (57.35 ml/min per 1.73 m 2 ; 95% CI, 53.18 to 61.52; P =0.1). There were no significant differences between groups in rates of delayed graft function, biopsy-proven acute rejection (BPAR), development of de novo donor-specific antibodies, or graft loss/death. Immunosuppression did not differ, and day 7 post-KTx plasma analyses showed approximately ten-fold lower TNF ( P <0.001) in IFX versus PLBO. BK viremia requiring IS change occurred more frequently in IFX (28.9%) versus PLBO (13.4%; P =0.004), with a strong trend toward higher rates of BKV nephropathy in IFX (13.3%) versus PLBO (4.9%; P =0.06). CONCLUSIONS: IFX induction therapy does not benefit recipients of kidney transplants from deceased donors on this IS regimen. Because the intervention unexpectedly increased rates of BK virus infections, our findings underscore the complexities of targeting peritransplant inflammation as a strategy to improve KTx outcomes.Clinical Trial registry name and registration number:clinicaltrials.gov (NCT02495077).

Machine perfusion in liver transplantation: recent advances and coming challenges.

PURPOSE OF REVIEW: Machine perfusion has been adopted into clinical practice in Europe since the mid-2010s and, more recently, in the United States (US) following approval of normothermic machine perfusion (NMP). We aim to review recent advances, provide discussion of potential future directions, and summarize challenges currently facing the field. RECENT FINDINGS: Both NMP and hypothermic-oxygenated perfusion (HOPE) improve overall outcomes after liver transplantation versus traditional static cold storage (SCS) and offer improved logistical flexibility. HOPE offers additional protection to the biliary system stemming from its' protection of mitochondria and lessening of ischemia-reperfusion injury. Normothermic regional perfusion (NRP) is touted to offer similar protective effects on the biliary system, though this has not been studied prospectively.The most critical question remaining is the optimal use cases for each of the three techniques (NMP, HOPE, and NRP), particularly as HOPE and NRP become more available in the US. There are additional questions regarding the most effective criteria for viability assessment and the true economic impact of these techniques. Finally, with each technique purported to allow well tolerated use of riskier grafts, there is an urgent need to define terminology for graft risk, as baseline population differences make comparison of current data challenging. SUMMARY: Machine perfusion is now widely available in all western countries and has become an essential tool in liver transplantation. Identification of the ideal technique for each graft, optimization of viability assessment, cost-effectiveness analyses, and proper definition of graft risk are the next steps to maximizing the utility of these powerful tools.

Linking donor-specific antibody generation with natural killer cells in antibody-mediated kidney graft rejection.

Natural killer (NK) cell infiltration of kidney allografts is a distinguishing feature of antibody-mediated rejection. Bailly et al. identify a distinct population of cytotoxic CD160+ interleukin-21 receptor+ CD56dimCD16bright NK cells that are uniquely found in the peripheral blood of donor-specific antibody-positive kidney transplant recipients and are present in kidney allografts with active antibody-mediated rejection. This population is implicated in a T follicular helper/interleukin-21/NK cell axis that links donor-specific antibody generation with graft-infiltrating NK cells in antibody-mediated rejection.

Autoantibodies against DNA topoisomerase I promote renal allograft rejection by increasing alloreactive T cell responses.

Antibodies reactive to self-antigens are an important component of posttransplant immune responses. The generation requirements and functions of autoantibodies, as well as the mechanisms of their influence on alloimmune responses, still remain to be determined. Our study investigated the contribution of autoimmunity during rejection of renal allografts. We have previously characterized a mouse model in which the acute rejection of a life-supporting kidney allograft is mediated by antibodies. At rejection, recipient sera screening against >4000 potential autoantigens revealed DNA topoisomerase I peptide 205-219 (TI-I205-219) as the most prominent epitope. Subsequent analysis showed TI-I205-219-reactive autoantibodies are induced in nonsensitized recipients of major histocompatibility complex-mismatched kidney allografts in a T cell-dependent manner. Immunization with TI-I205-219 broke self-tolerance, elicited TI-I205-219 immunoglobin G autoantibodies, and resulted in acute rejection of allogeneic but not syngeneic renal transplants. The graft loss was associated with increased priming of donor-reactive T cells but not with donor-specific alloantibodies elevation. Similarly, passive transfer of anti-TI-I205-219 sera following transplantation increased donor-reactive T cell activation with minimal effects on donor-specific alloantibody levels. The results identify DNA topoisomerase I as a novel self-antigen in transplant settings and demonstrate that autoantibodies enhance activation of donor-reactive T cells following renal transplantation.

Molecular Signature of Antibody-Mediated Chronic Vasculopathy in Heart Allografts in a Novel Mouse Model.

Cardiac allograft vasculopathy (CAV) limits the long-term success of heart transplants. Generation of donor-specific antibodies (DSAs) is associated with increased incidence of CAV clinically, but mechanisms underlying development of this pathology remain poorly understood. Major histocompatibility complex-mismatched A/J cardiac allografts in B6.CCR5-/- recipients have been reported to undergo acute rejection with little T-cell infiltration, but intense deposition of C4d in large vessels and capillaries of the graft accompanied by high titers of DSA. This model was modified to investigate mechanisms of antibody-mediated CAV by transplanting A/J hearts to B6.CCR5-/- CD8-/- mice that were treated with low doses of anti-CD4 monoclonal antibody to decrease T-cell-mediated graft injury and promote antibody-mediated injury. Although the mild inhibition of CD4 T cells extended allograft survival, the grafts developed CAV with intense C4d deposition and macrophage infiltration by 14 days after transplantation. Development of CAV correlated with recipient DSA titers. Transcriptomic analysis of microdissected allograft arteries identified the Notch ligand Dll4 as the most elevated transcript in CAV, associated with high versus low titers of DSA. More importantly, these analyses revealed a differential expression of transcripts in the CAV lesions compared with the matched apical tissue that lacks large arteries. In conclusion, these findings report a novel model of antibody-mediated CAV with the potential to facilitate further understanding of the molecular mechanisms promoting development of CAV.

A double-blind, placebo-controlled, randomized withdrawal trial of sarilumab for the treatment of glucocorticoid-dependent sarcoidosis.

OBJECTIVES: Effective steroid-sparing therapies for the treatment of sarcoidosis are lacking; interleukin-6 (IL-6) antagonists may reduce sarcoidosis disease activity. This study assessed the safety and efficacy of the IL-6 receptor antagonist, sarilumab, in subjects with glucocorticoid-dependent sarcoidosis. METHODS: This phase II, double-blind, placebo-controlled, randomized withdrawal trial enrolled 15 subjects with biopsy-proven sarcoidosis at Stanford University from November 2019 to September 2022. In Period 1, subjects were treated with open-label sarilumab 200mg subcutaneously every two weeks for 16 weeks, with predefined tapering of prednisone. Subjects who completed Period 1 without a sarcoidosis flare entered Period 2 and were randomized to continue sarilumab or to receive matching placebo for 12 weeks. Endpoints included flare-free survival, as well as changes in pulmonary function tests, chest imaging, patient reported outcomes, and laboratory values. RESULTS: Fifteen subjects were enrolled in the study (median age 57 years, 80% male, 73.3% White), and 10 subjects successfully completed Period 1. During Period 1, 4 of 15 subjects (26.7%) discontinued due to worsening of their sarcoidosis, and CT chest imaging worsened in 5 of 15 subjects (35.7%). During Period 2, 0 of 2 subjects in the sarilumab group and 1 of 8 subjects (12.5%) in the placebo group had a flare. Treatment with sarilumab 200 mg was generally well tolerated in subjects with sarcoidosis. CONCLUSION: In this double-blind, placebo-controlled, randomized withdrawal trial, a meaningful signal for improvement in subjects with sarcoidosis treated with sarilumab was not observed. Given the small numbers in this study, no definitive conclusions can be drawn. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04008069.

Macrophage-inducible C-type lectin activates B cells to promote T cell reconstitution in heart allograft recipients.

Diminishing homeostatic proliferation of memory T cells is essential for improving the efficacy of lymphoablation in transplant recipients. Our previous studies in a mouse heart transplantation model established that B lymphocytes secreting proinflammatory cytokines are critical for T cell recovery after lymphoablation. The goal of the current study was to identify mediators of B cell activation following lymphoablation in allograft recipients. Transcriptome analysis revealed that macrophage-inducible C-type lectin (Mincle, Clec4e) expression is up-regulated in B cells from heart allograft recipients treated with murine anti-thymocyte globulin (mATG). Recipient Mincle deficiency diminishes B cell production of pro-inflammatory cytokines and impairs T lymphocyte reconstitution. Mixed bone marrow chimeras lacking Mincle only in B lymphocytes have similar defects in T cell recovery. Conversely, treatment with a synthetic Mincle ligand enhances T cell reconstitution after lymphoablation in non-transplanted mice. Treatment with agonistic CD40 mAb facilitates T cell reconstitution in CD4 T cell-depleted, but not in Mincle-deficient, recipients indicating that CD40 signaling induces T cell proliferation via a Mincle-dependent pathway. These findings are the first to identify an important function of B cell Mincle as a sensor of damage-associated molecular patterns released by the graft and demonstrate its role in clinically relevant settings of organ transplantation.

C1q as a potential tolerogenic therapeutic in transplantation.

In 1963, Lepow and colleagues resolved C1, the first component of the classical pathway, into three components, which they named C1q, C1r, and C1s. All three of these components were demonstrated to be involved in causing hemolysis in vitro. For over 30 years after that seminal discovery, the primary function attributed to C1q was as part of the C1 complex that initiated the classical pathway of the complement cascade. Then, a series of papers reported that isolated C1q could bind to apoptotic cells and facilitate their clearance by macrophages. Since then, rheumatologists have recognized that C1q is an important pattern recognition receptor (PRR) that diverts autoantigen containing extracellular vesicles from immune recognition. This critical function of C1q as a regulator of immune recognition has been largely overlooked in transplantation. Now that extracellular vesicles released from transplants have been identified as a major agent of immune recognition, it is logical to consider the potential impact of C1q on modulating the delivery of allogeneic extracellular vesicles to antigen presenting cells. This concept has clinical implications in the possible use of C1q or a derivative as a biological therapeutic to down-modulate immune responses to transplants.

Continuous function of 80 primary renal allografts for 30-47 years with maintenance prednisone and azathioprine/mycophenolate mofetil therapy: A clinical mosaic of long-term successes.

Eighty primary renal allograft recipients, 61 living-related and 19 deceased donor, transplanted from 1963 through 1984 had continuous graft function for 30-47 years. They were treated with three different early immunosuppression programs (1963-1970: thymectomy, splenectomy, high oral prednisone; 1971-1979: divided-dose intravenous methylprednisolone; and 1980-1984: antilymphocyte globulin) each with maintenance prednisone and azathioprine, and no calcineurin inhibitor. Long-term treatment often included the anti-platelet medication, dipyridamole. Although both recipient and donor ages were young (27.2 ± 9.5 and 33.1 ± 12.0 years, respectively), six recipients with a parent donor had >40-year success. At 35 years, death-censored graft survival was 85.3% and death with a functioning graft 84.2%; overall graft survival was 69.5% (Kaplan-Meier estimate). Biopsy-documented early acute cellular and highly probable antibody-mediated rejections were reversed with divided-dose intravenous methylprednisolone. Complications are detailed in an integrated timeline. Hypogammaglobulinemia identified after 20 years doubled the infection rate. An association between a monoclonal gammopathy of undetermined significance and non-plasma-cell malignancies was identified. Twenty-seven azathioprine-treated patients tested after 37 years had extremely low levels of T1/T2 B lymphocytes representing a "low immunosuppression state of allograft acceptance (LISAA)". The lifetime achievements of these patients following a single renal allograft and low-dose maintenance immunosuppression are remarkable. Their success evolved as a clinical mosaic.

Endogenous memory T cells with donor-reactivity: early post-transplant mediators of acute graft injury in unsensitized recipients.

The pretransplant presence of endogenous donor-reactive memory T cells is an established risk factor for acute rejection and poorer transplant outcomes. A major source of these memory T cells in unsensitized recipients is heterologously generated memory T cells expressing reactivity to donor allogeneic MHC molecules. Multiple clinical studies have shown that the pretransplant presence of high numbers of circulating endogenous donor-reactive memory T cells correlates with higher incidence of acute rejection and decreased graft function during the first-year post-transplant. These findings have spurred investigation in preclinical models to better understand mechanisms underlying endogenous donor-reactive memory T-cell-mediated allograft injury in unsensitized graft recipients. These studies have led to the identification of unique mechanisms underlying the activation of these memory T cells within allografts at early times after transplant. In particular, optimal activation to mediate acute allograft injury is dependent on the intensity of ischaemia-reperfusion injury. Therapeutic strategies directed at the recruitment and activation of endogenous donor-reactive memory T cells are effective in attenuating acute injury in allografts experiencing increased ischaemia-reperfusion injury in preclinical models and should be translatable to clinical transplantation.

Neutrophil Cathepsin G Regulates Dendritic Cell Production of IL-12 during Development of CD4 T Cell Responses to Antigens in the Skin.

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to hapten skin sensitization and challenge. Sensitization of wild-type (WT) mice induces hapten-reactive effector CD8 T cells producing IFN-γ and IL-17- and IL-4-producing CD4 T cells that cannot mediate CHS. Although CXCR2-dependent Ly6G+ (neutrophil) cell recruitment into hapten-challenged skin is required to direct effector CD8 T cell infiltration into the challenge site to elicit CHS, 2,4-dinitrofluorobenezene (DNFB) sensitization of CXCR2-/- mice and neutrophil-depleted WT mice induced both hapten-reactive CD4 and CD8 T cells producing IFN-γ and IL-17. CD4 T cell-mediated CHS responses were not generated during DNFB sensitization of neutrophil-depleted WT mice treated with anti-IL-12 mAb or neutrophil-depleted IL-12-/- mice. Neutrophil depletion during DNFB sensitization of WT mice markedly increased IL-12-producing hapten-primed dendritic cell numbers in the skin-draining lymph nodes. Sensitization of mice lacking the neutrophil serine protease cathepsin G (CG)-induced hapten-reactive CD4 and CD8 T cells producing IFN-γ and IL-17 with elevated and elongated CHS responses to DNFB challenge. Induction of CHS effector CD4 T cells producing IFN-γ in neutrophil-depleted WT mice was eliminated by s.c. injection of active, but not inactivated, CG during sensitization. Thus, hapten skin sensitization induces neutrophil release of CG that systemically inhibits hapten-presenting dendritic cell production of IL-12 and the development of hapten-reactive CD4 T cells to IFN-γ-producing CHS effector cells.

Developmental loss, but not pharmacological suppression, of renal carbonic anhydrase 2 results in pyelonephritis susceptibility.

Carbonic anhydrase II knockout (Car2-/-) mice have depleted numbers of renal intercalated cells, which are increasingly recognized to be innate immune effectors. We compared pyelonephritis susceptibility following reciprocal renal transplantations between Car2-/- and wild-type mice. We examined the effect of pharmacological CA suppression using acetazolamide in an experimental murine model of urinary tract infection. Car2-/- versus wild-type mice were compared for differences in renal innate immunity. In our transplant scheme, mice lacking CA-II in the kidney had increased pyelonephritis risk. Mice treated with acetazolamide had lower kidney bacterial burdens at 6 h postinfection, which appeared to be due to tubular flow from diuresis because comparable results were obtained when furosemide was substituted for acetazolamide. Isolated Car2-/- kidney cells enriched for intercalated cells demonstrated altered intercalated cell innate immune gene expression, notably increased calgizzarin and insulin receptor expression. Intercalated cell number and function along with renal tubular flow are determinants of pyelonephritis risk.

Early T cell infiltration is modulated by programed cell death-1 protein and its ligand (PD-1/PD-L1) interactions in murine kidney transplants.

Allogeneic transplants elicit dynamic T cell responses that are modulated by positive and negative co-stimulatory receptors. Understanding mechanisms that intrinsically modulate the immune responses to transplants is vital to develop rational treatment for rejection. Here, we have investigated the impact of programed cell death-1 (PD-1) protein, a negative co-stimulatory receptor, on the rejection of MHC incompatible kidney transplants in mice. T cells were found to rapidly infiltrate the kidneys of A/J mice transplanted to C57BL/6 mice, which peaked at six days and decline by day 14. The T cells primarily encircled tubules with limited infiltration of the tubular epithelium. Lipocalin 2 (LCN2), a marker of tubular injury, also peaked in the urine at day six and then declined. Notably, flow cytometry demonstrated that most of the T cells expressed PD-1 (over 90% of CD8 and about 75% of CD4 cells) at day six. Administration of blocking antibody to PD-L1, the ligand for PD-1, before day six increased T cell infiltrates and urinary LCN2, causing terminal acute rejection. In contrast, blocking PD-1/PD-L1 interactions after day six caused only a transient increase in urinary LCN2. Depleting CD4 and CD8 T cells virtually eliminated LCN2 in the urine in support of T cells injuring tubules. Thus, our data indicate that PD-1/PD-L1 interactions are not just related to chronic antigenic stimulation of T cells but are critical for the regulation of acute T cell responses to renal transplants.

B cell-derived IL-1ß and IL-6 drive T cell reconstitution following lymphoablation.

Understanding the mechanisms of T cell homeostatic expansion is crucial for clinical applications of lymphoablative therapies. We previously established that T cell recovery in mouse heart allograft recipients treated with anti-thymocyte globulin (mATG) critically depends on B cells and is mediated by B cell-derived soluble factors. B cell production of interleukin (IL)-1ß and IL-6 is markedly upregulated after heart allotransplantation and lymphoablation. Neutralizing IL-1ß or IL-6 with mAb or the use of recipients lacking mature IL-1ß, IL-6, IL-1R, MyD88, or IL-6R impair CD4+ and CD8+ T cell recovery and significantly enhance the graft-prolonging efficacy of lymphoablation. Adoptive co-transfer experiments demonstrate a direct effect of IL-6 but not IL-1ß on T lymphocytes. Furthermore, B cells incapable of IL-1ß or IL-6 production have diminished capacity to mediate T cell reconstitution and initiate heart allograft rejection upon adoptive transfer into mATG treated B cell deficient recipients. These findings reveal the essential role of B cell-derived IL-1ß and IL-6 during homeostatic T cell expansion in a clinically relevant model of lymphoablation.

Antibody-induced vascular inflammation skews infiltrating macrophages to a novel remodeling phenotype in a model of transplant rejection.

HLA donor-specific antibodies (DSAs) binding to vascular endothelial cells of the allograft trigger inflammation, vessel injury, and antibody-mediated rejection (AMR). Accumulation of intragraft-recipient macrophages is a histological characteristic of AMR, which portends worse outcome. HLA class I (HLA I) DSAs enhance monocyte recruitment by activating endothelial cells and engaging FcγRs, but the DSA-activated donor endothelial influence on macrophage differentiation is unknown. In this study, we explored the consequence of DSA-activated endothelium on infiltrating monocyte differentiation. Here we show that cardiac allografts from murine recipients treated with MHC I DSA upregulated genes related to monocyte transmigration and Fc receptor stimulation. Human monocytes co-cultured with HLA I IgG-stimulated primary human endothelium promoted monocyte differentiation into CD68+ CD206+ CD163+ macrophages (M(HLA I IgG)), whereas HLA I F(ab')2 stimulated endothelium solely induced higher CD206 (M(HLA I F(ab')2 )). Both macrophage subtypes exhibited significant changes in discrete cytokines/chemokines and unique gene expression profiles. Cross-comparison of gene transcripts between murine DSA-treated cardiac allografts and human co-cultured macrophages identified overlapping genes. These findings uncover the role of HLA I DSA-activated endothelium in monocyte differentiation, and point to a novel, remodeling phenotype of infiltrating macrophages that may contribute to vascular injury.