Longitudinal immune profile reveals reduced function of pro-inflammatory monocytes with age following kidney transplantation

Toxicity of immunosuppression, notably the risk of infection, increases with age. However, the dynamic changes in innate immune response following transplantation are unclear. Based on recent observations, we hypothesized that pro-inflammatory capacity would decrease with age. We analyzed approximately 300 PBMC samples collected longitudinally in 45 de novo, adult kidney recipients and performed detailed phenotypic and functional profiling of monocytes and T cell subsets. Inflammatory response to TLR4 stimulation and indirect allostimulation using mismatched HLA peptides were assessed. In patients aged ≥56 years, TNF-α production by intermediate monocytes was similar to that in younger patients early posttransplant, but diminished substantially later. Adjusted analyses suggested that this was not attributable to confounding factors. In contrast, the alloimmune response to HLA peptides measured by IFN-γ in CD4⁺ T cells and TNF-α in monocytes was stable over time, but was low in older recipients. Measurement of CD80-86 surface expression revealed no signal for a lower costimulation capacity of APCs. These results suggest that older recipients have a reduced function of their innate pro-inflammatory immune cells posttransplant while maintaining a stable, low alloimmune response over time. The effect of reduced immunosuppressant doses on preventing this phenomenon needs to be clarified.     

Neuro‐immune interactions in inflammation and host defense: Implications for transplantation

Sensory and autonomic neurons of the peripheral nervous system (PNS) play a critical role in regulating the immune system during tissue inflammation and host defense. Recent studies have identified the molecular mechanisms underlying the bidirectional communication between the nervous system and the immune system. Here, we highlight the studies that demonstrate the importance of the neuro‐immune interactions in health and disease. Nociceptor sensory neurons detect immune mediators to produce pain, and release neuropeptides that act on the immune system to regulate inflammation. In parallel, neural reflex circuits including the vagus nerve‐based inflammatory reflex are physiological regulators of inflammatory responses and cytokine production. In transplantation, neuro‐immune communication could significantly impact the processes of host‐pathogen defense, organ rejection, and wound healing. Emerging approaches to target the PNS such as bioelectronics could be useful in improving the outcome of transplantation. Therefore, understanding how the nervous system shapes the immune response could have important therapeutic ramifications for transplantation medicine     

Discovery and validation of a novel blood‐based molecular biomarker of rejection following liver transplantation

Noninvasive biomarker profiles of acute rejection (AR) could affect the management of liver transplant (LT) recipients. Peripheral blood was collected following LT for discovery (Northwestern University [NU]) and validation (National Institute of Allergy and Infectious Diseases Clinical Trials in Organ Transplantation [CTOT]‐14 study). Blood gene profiling was paired with biopsies showing AR or ADNR (acute dysfunction no rejection) as well as stable graft function samples (Transplant eXcellent—TX). CTOT‐14 subjects had serial collections prior to AR, ADNR, TX, and after AR treatment. NU cohort gene expression (46 AR, 45 TX) was analyzed using random forest models to generate a classifier training set (36 gene probe) distinguishing AR vs TX (area under the curve 0.92). The algorithm and threshold were locked and tested on the CTOT‐14 validation cohort (14 AR, 50 TX), yielding an accuracy of 0.77, sensitivity 0.57, specificity 0.82, positive predictive value (PPV) 0.47, and negative predictive value (NPV) 0.87 for AR vs TX. The probability score line slopes were positive preceding AR, and negative preceding TX and non‐AR (TX + ADNR) (P ≤ .001) and following AR treatment. In conclusion, we have developed a blood biomarker diagnostic for AR that can be detected prior to AR‐associated graft injury as well a normal graft function (non‐AR). Further studies are needed to evaluate its utility in precision‐guided immunosuppression optimization following LT.     

Outside‐in HLA class I signaling regulates ICAM‐1 clustering and endothelial cell‐monocyte interactions via mTOR in transplant antibody‐mediated rejection

Antibody‐mediated rejection (AMR) resulting in transplant allograft vasculopathy (TAV) is the major obstacle for long‐term survival of solid organ transplants. AMR is caused by donor‐specific antibodies to HLA, which contribute to TAV by initiating outside‐in signaling transduction pathways that elicit monocyte recruitment to activated endothelium. Mechanistic target of rapamycin (mTOR) inhibitors can attenuate TAV; therefore, we sought to understand the mechanistic underpinnings of mTOR signaling in HLA class I Ab–mediated endothelial cell activation and monocyte recruitment. We used an in vitro model to assess monocyte binding to HLA I Ab–activated endothelial cells and found mTOR inhibition reduced ezrin/radixin/moesin (ERM) phosphorylation, intercellular adhesion molecule 1 (ICAM‐1) clustering, and monocyte firm adhesion to HLA I Ab–activated endothelium. Further, in a mouse model of AMR, in which C57BL/6. RAG1^?/? recipients of BALB/c cardiac allografts were passively transferred with donor‐specific MHC I antibodies, mTOR inhibition significantly reduced vascular injury, ERM phosphorylation, and macrophage infiltration of the allograft. Taken together, these studies indicate mTOR inhibition suppresses ERM phosphorylation in endothelial cells, which impedes ICAM‐1 clustering in response to HLA class I Ab and prevents macrophage infiltration into cardiac allografts. These findings indicate a novel therapeutic application for mTOR inhibitors to disrupt endothelial cell‐monocyte interactions during AMR.     

Immune Exhaustion and Transplantation

Exhaustion of lymphocyte function through chronic exposure to a high load of foreign antigen is well established for chronic viral infection and antitumor immunity and has been found to be associated with a distinct molecular program and characteristic cell surface phenotype. Although exhaustion has most commonly been studied in the context of CD8 viral responses, recent studies indicate that chronic antigen exposure may affect B cells, NK cells and CD4 T cells in a parallel manner. Limited information is available regarding the extent of lymphocyte exhaustion development in the transplant setting and its impact on anti–graft alloreactivity. By analogy to the persistence of a foreign virus, the large mass of alloantigen presented by an allograft in chronic residence could provide an ideal setting for exhausting donor‐reactive T cells. The extent of T cell exhaustion occurring with various allografts, the kinetics of its development, whether exhaustion is influenced positively or negatively by different immunosuppressants, and the impact of exhaustion on graft survival and tolerance development remains a fertile area for investigation. Harnessing or encouraging the natural processes of exhaustion may provide a novel means to promote graft survival and transplantation tolerance.     

Reversing donor‐specific antibody responses and antibody‐mediated rejection with bortezomib and belatacept in mice and kidney transplant recipients

Active antibody‐mediated rejection (AMR) is a potentially devastating complication and consistently effective treatment remains elusive. We hypothesized that the reversal of acute AMR requires rapid elimination of antibody‐secreting plasma cells (PC) with a proteasome inhibitor, bortezomib, followed by the sustained inhibition of PC generation with CTLA4‐Ig or belatacept (B/B). We show in mice that B/B therapy selectively depleted mature PC producing donor‐specific antibodies (DSA) and reduced DSA, when administered after primary and secondary DSA responses had been established. A pilot investigation was initiated to treat six consecutive patients with active AMR with B/B. Compassionate use of this regimen was initiated for the first patient who developed early, severe acute AMR that did not respond to steroids, plasmapheresis, and intravenous immunoglobulin after his third kidney transplant. B/B treatment resulted in a rapid reversal of AMR, leading us to treat five additional patients who also resolved their acute AMR episode and had sustained disappearance of circulating DSA for ≤30 months. This study provides a proof‐of‐principle demonstration that mouse models can identify mechanistically rational therapies for the clinic. Follow‐up investigations with a more stringent clinical design are warranted to test whether B/B improves on the standard of care for the treatment of acute AMR.     

Anti‐CD40 antibody 2C10 binds to a conformational epitope at the CD40‐CD154 interface that is conserved among primate species

The antagonistic anti‐CD40 antibody, 2C10, and its recombinant primate derivative, 2C10R4, are potent immunosuppressive antibodies whose utility in allo‐ and xenotransplantation have been demonstrated in nonhuman primate studies. In this study, we defined the 2C10 binding epitope and found only slight differences in affinity of 2C10 for CD40 derived from four primate species. Staining of truncation mutants mapped the 2C10 binding epitope to the N‐terminal portion of CD40. Alanine scanning mutagenesis of the first 60 residues in the CD40 ectodomain highlighted key amino acids important for binding of 2C10 and for binding of the noncross‐blocking anti‐CD40 antibodies 3A8 and 5D12. All four 2C10‐binding residues defined by mutagenesis clustered near the membrane‐distal tip of CD40 and partially overlap the CD154 binding surface. In contrast, the overlapping 3A8 and 5D12 epitopes map to an opposing surface away from the CD154 binding domain. This biochemical characterization of 2C10 confirms the validity of nonhuman primate studies in the translation of this therapeutic antibody and provides insight its mechanism of action.     

Trained immunity in organ transplantation

Consistent induction of donor‐specific unresponsiveness in the absence of continuous immunosuppressive therapy and toxic effects remains a difficult task in clinical organ transplantation. Transplant immunologists have developed numerous experimental treatments that target antigen‐presentation (signal 1), costimulation (signal 2), and cytokine production (signal 3) to establish transplantation tolerance. While promising results have been obtained using therapeutic approaches that predominantly target the adaptive immune response, the long‐term graft survival rates remain suboptimal. This suggests the existence of unrecognized allograft rejection mechanisms that contribute to organ failure. We postulate that trained immunity stimulatory pathways are critical to the immune response that mediates graft loss. Trained immunity is a recently discovered functional program of the innate immune system, which is characterized by nonpermanent epigenetic and metabolic reprogramming of macrophages. Since trained macrophages upregulate costimulatory molecules (signal 2) and produce pro‐inflammatory cytokines (signal 3), they contribute to potent graft reactive immune responses and organ transplant rejection. In this review, we summarize the detrimental effects of trained immunity in the context of organ transplantation and describe pathways that induce macrophage training associated with graft rejection.     

DEPTOR modulates activation responses in CD4+ T cells and enhances immunoregulation following transplantation

DEPTOR is an evolutionarily conserved cell‐intrinsic binding partner of mTOR that functions as a negative regulator of signaling responses. In this study, we show that DEPTOR is expressed within CD4⁺ T cells, and we observed that its relative level of expression modulates differentiation as well as glucose utilization within CD4⁺ T effectors in vitro. Using knock‐in mice, we also find that induced expression of DEPTOR within CD4⁺ T regulatory cells stabilizes Foxp3 expression, shifts metabolism toward oxidative phosphorylation, and increases survival and suppressive function. In vivo, fully MHC mismatched cardiac allograft survival is significantly prolonged in knock‐in recipients and sustained recipient expression of DEPTOR in combination with costimulatory blockade induces long‐term graft survival. Furthermore, we show that the induced expression of DEPTOR in CD4⁺ T effectors fails to inhibit acute allograft rejection. Rather, prolonged survival is dominantly mediated via induced expression and function of DEPTOR within recipient CD4⁺ T regulatory cells. These collective findings identify DEPTOR as a novel protein that functions in CD4⁺ T cells to augment immunoregulation in vitro and in vivo.     

C5aR1 regulates migration of suppressive myeloid cells required for costimulatory blockade‐induced murine allograft survival

Costimulatory blockade‐induced murine cardiac allograft survival requires intragraft accumulation of CD11b⁺Ly6C^loLy6G^? regulatory myeloid cells (Mregs) that expand regulatory T cells (Tregs) and suppress effector T cells (Teffs). We previously showed that C5a receptor (C5aR1) signaling on T cells activates Teffs and inhibits Tregs, but whether and/or how C5aR1 affects Mregs required for transplant survival is unknown. Although BALB/c hearts survived >60 days in anti‐CD154 (MR1)‐treated or cytotoxic T‐lymphocyte associated protein 4 (CTLA4)‐Ig–treated wild‐type (WT) recipients, they were rejected at ~30 days in MR1‐treated or CTLA4‐Ig–treated recipients selectively deficient in C5aR1 restricted to myeloid cells (C5ar1^fl/flxLysM‐Cre). This accelerated rejection was associated with ~2‐fold more donor‐reactive T cells and ~40% less expansion of donor‐reactive Tregs. Analysis of graft‐infiltrating mononuclear cells on posttransplant day 6 revealed fewer Ly6C^lo monocytes in C5ar1^fl/flxLysM‐Cre recipients. Expression profiling of intragraft Ly6C^lo monocytes showed that C5aR1 deficiency downregulated genes related to migration/locomotion without changes in genes associated with suppressive function. Cotransfer of C5ar1^fl/fl and C5ar1^fl/flxLysM‐Cre myeloid cells into MR1‐treated allograft recipients resulted in less accumulation of C5ar1^?/? cells within the allografts, and in vitro assays confirmed that Ly6C^hi myeloid cells migrate to C5a/C5aR1‐initiated signals. Together, our results newly link myeloid cell–expressed C5aR1 to intragraft accumulation of myeloid cells required for prolongation of heart transplant survival induced by costimulatory blockade.     

Continuous Acquisition of MHC:Peptide Complexes by Recipient Cells Contributes to the Generation of Anti‐Graft CD8+ T Cell Immunity

Understanding the evolution of the direct and indirect pathways of allorecognition following tissue transplantation is essential in the design of tolerance‐promoting protocols. On the basis that donor bone marrow–derived antigen‐presenting cells are eliminated within days of transplantation, it has been argued that the indirect response represents the major threat to long‐term transplant survival, and is consequently the key target for regulation. However, the detection of MHC transfer between cells, and particularly the capture of MHC:peptide complexes by dendritic cells (DCs), led us to propose a third, semidirect, pathway of MHC allorecognition. Persistence of this pathway would lead to sustained activation of direct‐pathway T cells, arguably persisting for the life of the transplant. In this study, we focused on the contribution of acquired MHC‐class I on recipient DCs during the life span of a skin graft. We observed that MHC‐class I acquisition by recipient DCs occurs for at least 1 month following transplantation and may be the main source of alloantigen that drives CD8⁺ cytotoxic T cell responses. In addition, acquired MHC‐class I:peptide complexes stimulate T cell responses in vivo, further emphasizing the need to regulate both pathways to induce indefinite survival of the graft.     

Circulating T follicular helper cells are a biomarker of humoral alloreactivity and predict donor‐specific antibody formation after transplantation

Donor‐specific antibodies (DSAs) contribute to renal allograft loss. However, biomarkers to guide clinical management of DSA posttransplant or detect humoral alloimmune responses before alloantibodies develop are not available. Circulating T follicular helper (cTfh) cells are CD4⁺CXCR5⁺ Tfh‐like cells in the blood that have been associated with alloantibodies in transplant recipients, but whether they precede antibody formation for their evaluation as a predictive biomarker in transplant is unknown. To evaluate the ability of cTfh cells to predict DSA, we used murine transplant models to determine the temporal relationship between cTfh cells, germinal center formation, and DSA development. We observed that donor‐reactive CD4⁺CXCR5⁺ cTfh cells expand after allotransplant. These cTfh cells were equivalent to graft‐draining lymph node‐derived Tfh cells in their ability to provide B cell help for antibody production. cTfh cell expansion and differentiation into ICOS⁺PD‐1⁺ cells temporally correlated with germinal center alloreactivity and preceded the generation of DSAs in instances of modified and unmodified alloantibody formation. Importantly, delayed costimulation blockade initiated after the detection of ICOS⁺PD‐1⁺ cTfh cells prevented DSAs. These findings suggest that cTfh cells could serve as a biomarker for humoral alloreactivity before the detection of alloantibodies and inform therapeutic approaches to prevent DSAs.     

Prospective Analyses of Circulating B Cell Subsets in ABO‐Compatible and ABO‐Incompatible Kidney Transplant Recipients

Immunosuppressive strategies applied in renal transplantation traditionally focus on T cell inhibition. B cells were mainly examined in the context of antibody‐mediated rejection, whereas the impact of antibody‐independent B cell functions has only recently entered the field of transplantation. Similar to T cells, distinct B cell subsets can enhance or inhibit immune responses. In this study, we prospectively analyzed the evolution of B cell subsets in the peripheral blood of ABO‐compatible (n = 27) and ABO‐incompatible (n = 10) renal transplant recipients. Activated B cells were transiently decreased and plasmablasts were permanently decreased in patients without signs of rejection throughout the first year. In patients with histologically confirmed renal allograft rejection, activated B cells and plasmablasts were significantly elevated on day 365. Rituximab treatment in ABO‐incompatible patients resulted in long‐lasting B cell depletion and in a naïve phenotype of repopulating B cells 1 year following transplantation. Acute allograft rejection was correlated with an increase of activated B cells and plasmablasts and with a significant reduction of regulatory B cell subsets. Our study demonstrates the remarkable effects of standard immunosuppression on circulating B cell subsets. Furthermore, the B cell compartment was significantly altered in rejecting patients. A specific targeting of deleterious B cell subsets could be of clinical benefit in renal transplantation.     

Selective Targeting of High‐Affinity LFA‐1 Does Not Augment Costimulation Blockade in a Nonhuman Primate Renal Transplantation Model

Costimulation blockade (CoB) via belatacept is a lower‐morbidity alternative to calcineurin inhibitor (CNI)‐based immunosuppression. However, it has higher rates of early acute rejection. These early rejections are mediated in part by memory T cells, which have reduced dependence on the pathway targeted by belatacept and increased adhesion molecule expression. One such molecule is leukocyte function antigen (LFA)‐1. LFA‐1 exists in two forms: a commonly expressed, low‐affinity form and a transient, high‐affinity form, expressed only during activation. We have shown that antibodies reactive with LFA‐1 regardless of its configuration are effective in eliminating memory T cells but at the cost of impaired protective immunity. Here we test two novel agents, leukotoxin A and AL‐579, each of which targets the high‐affinity form of LFA‐1, to determine whether this more precise targeting prevents belatacept‐resistant rejection. Despite evidence of ex vivo and in vivo ligand‐specific activity, neither agent when combined with belatacept proved superior to belatacept monotherapy. Leukotoxin A approached a ceiling of toxicity before efficacy, while AL‐579 failed to significantly alter the peripheral immune response. These data, and prior studies, suggest that LFA‐1 blockade may not be a suitable adjuvant agent for CoB‐resistant rejection.     

Long‐Term Persistence of Donor Alveolar Macrophages in Human Lung Transplant Recipients That Influences Donor‐Specific Immune Responses

Steady‐state alveolar macrophages (AMs) are long‐lived lung‐resident macrophages with sentinel function. Evidence suggests that AM precursors originate during embryogenesis and populate lungs without replenishment by circulating leukocytes. However, their presence and persistence are unclear following human lung transplantation (LTx). Our goal was to examine donor AM longevity and evaluate whether AMs of recipient origin seed the transplanted lungs. Origin of AMs was accessed using donor–recipient HLA mismatches. We demonstrate that 94–100% of AMs present in bronchoalveolar lavage (BAL) were donor derived and, importantly, AMs of recipient origin were not detected. Further, analysis of BAL cells up to 3.5 years post‐LTx revealed that the majority of AMs (>87%) was donor derived. Elicitation of de novo donor‐specific antibody (DSA) is a major post‐LTx complication and a risk factor for development of chronic rejection. The donor AMs responded to anti‐HLA framework antibody (Ab) with secretion of inflammatory cytokines. Further, in an experimental murine model, we demonstrate that adoptive transfer of allogeneic AMs stimulated humoral and cellular immune responses to alloantigen and lung‐associated self‐antigens and led to bronchiolar obstruction. Therefore, donor‐derived AMs play an essential role in the DSA‐induced inflammatory cascade leading to obliterative airway disease of the transplanted lungs.     

B cell–derived IL‐1β 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)‐1β and IL‐6 is markedly upregulated after heart allotransplantation and lymphoablation. Neutralizing IL‐1β or IL‐6 with mAb or the use of recipients lacking mature IL‐1β, 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‐1β on T lymphocytes. Furthermore, B cells incapable of IL‐1β 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‐1β and IL‐6 during homeostatic T cell expansion in a clinically relevant model of lymphoablation.