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Objectives: To characterize and quantify accumulating immunologic alterations, pre and postoperatively in patients undergoing elective surgical procedures., Background: Elective surgery is an anticipatable, controlled human injury. Although the human response to injury is generally stereotyped, individual variability exists. This makes surgical outcomes less predictable, even after standardized procedures, and may provoke complications in patients unable to compensate for their injury. One potential source of variation is found in immune cell maturation, with phenotypic changes dependent on an individual's unique, lifelong response to environmental antigens., Methods: We enrolled 248 patients in a prospective trial facilitating comprehensive biospecimen and clinical data collection in patients scheduled to undergo elective surgery. Peripheral blood was collected preoperatively, and immediately on return to the postanesthesia care unit. Postoperative complications that occurred within 30 days after surgery were captured., Results: As this was an elective surgical cohort, outcomes were generally favorable. With a median follow-up of 6 months, the overall survival at 30 days was 100%. However, 20.5% of the cohort experienced a postoperative complication (infection, readmission, or system dysfunction). We identified substantial heterogeneity of immune senescence and terminal differentiation phenotypes in surgical patients. More importantly, phenotypes indicating increased T-cell maturation and senescence were associated with postoperative complications and were evident preoperatively., Conclusions: The baseline immune repertoire may define an immune signature of resilience to surgical injury and help predict risk for surgical complications., Competing Interests: The authors report no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Thoracic injury can cause impairment of lung function leading to respiratory complications such as pneumonia (PNA). There is increasing evidence that central memory T cells of the adaptive immune system play a key role in pulmonary immunity. We sought to explore whether assessment of cell phenotypes using flow cytometry (FCM) could be used to identify pulmonary infection after thoracic trauma., Methods: We prospectively studied trauma patients with thoracic injuries who survived >48 hours at a Level 1 trauma center from 2014 to 2020. Clinical and FCM data from serum samples collected within 24 hours of admission were considered as potential variables. Random forest and logistic regression models were developed to estimate the risk of hospital-acquired and ventilator-associated PNA. Variables were selected using backwards elimination, and models were internally validated with leave-one-out., Results: Seventy patients with thoracic injuries were included (median age, 35 years [interquartile range (IQR), 25.25-51 years]; 62.9% [44 of 70] male, 61.4% [42 of 70] blunt trauma). The most common injuries included rib fractures (52 of 70 [74.3%]) and pulmonary contusions (26 of 70 [37%]). The incidence of PNA was 14 of 70 (20%). Median Injury Severity Score was similar for patients with and without PNA (30.5 [IQR, 22.6-39.3] vs. 26.5 [IQR, 21.6-33.3]). The final random forest model selected three variables (Acute Physiology and Chronic Health Evaluation score, highest pulse rate in first 24 hours, and frequency of CD4 + central memory cells) that identified PNA with an area under the curve of 0.93, sensitivity of 0.91, and specificity of 0.88. A logistic regression with the same features had an area under the curve of 0.86, sensitivity of 0.76, and specificity of 0.85., Conclusion: Clinical and FCM data have diagnostic utility in the early identification of patients at risk of nosocomial PNA following thoracic injury. Signs of physiologic stress and lower frequency of central memory cells appear to be associated with higher rates of PNA after thoracic trauma., Level of Evidence: Diagnostic Test/Criteria; Level IV., (Copyright © 2023 Written work prepared by employees of the Federal Government as part of their official duties is, under the U.S. Copyright Act, a "œwork of the United States Government"œ for which copyright protection under Title 17 of the United States Code is not available. As such, copyright does not extend to the contributions of employees of the Federal Government.)

Background: Malnutrition, including obesity and undernutrition, among children is increasing in prevalence and is common among children on renal replacement therapy. The effect of malnutrition on the pre-transplant immune system and how the pediatric immune system responds to the insult of both immunosuppression and allotransplantation is unknown. We examined the relationship of nutritional status with post-transplant outcomes and characterized the peripheral immune cell phenotypes of children from the Immune Development of Pediatric Transplant (IMPACT) study., Methods: Ninety-eight patients from the IMPACT study were classified as having obesity, undernutrition, or normal nutrition-based pre-transplant measurements. Incidence of infectious and alloimmune outcomes at 1-year post-transplantation was compared between nutritional groups using Gray's test and Fine-Gray subdistribution hazards model. Event-free survival was estimated by Kaplan-Meier method and compared between groups. Differences in immune cell subsets between nutritional groups over time were determined using generalized estimating equations accounting for the correlation between repeated measurements., Results: We did not observe that nutritional status was associated with infectious or alloimmune events or event-free survival post-transplant. We demonstrated that children with obesity had distinct T-and B-cell signatures relative to those with undernutrition and normal nutrition, even when controlling for immunosuppression. Children with obesity had a lower frequency of CD8 Tnaive cells 9-month post-transplant (p < .001), a higher frequency of CD4 CD57 + PD1- T cells, and lower frequencies of CD57-PD1+ CD8 and CD57-PD1- CD8 T cells at 12-month transplant (p < .05 for all)., Conclusions: Children with obesity have distinct immunophenotypes that may influence the tailoring of immunosuppression., (© 2022 Wiley Periodicals LLC.)

Background: An emerging body of literature supports the role of individualized prognostic tools to guide the management of patients after trauma. The aim of this study was to develop advanced modeling tools from multidimensional data sources, including immunological analytes and clinical and administrative data, to predict outcomes in trauma patients., Methods: This was a prospective study of trauma patients at Level 1 centers from 2015 to 2019. Clinical, flow cytometry, and serum cytokine data were collected within 48 hours of admission. Sparse logistic regression models were developed, jointly selecting predictors and estimating the risk of ventilator-associated pneumonia, acute kidney injury, complicated disposition (death, rehabilitation, or nursing facility), and return to the operating room. Model parameters (regularization controlling model sparsity) and performance estimation were obtained via nested leave-one-out cross-validation., Results: A total of 179 patients were included. The incidences of ventilator-associated pneumonia, acute kidney injury, complicated disposition, and return to the operating room were 17.7%, 28.8%, 22.5%, and 12.3%, respectively. Regarding extensive resource use, 30.7% of patients had prolonged intensive care unit stay, 73.2% had prolonged length of stay, and 23.5% had need for prolonged ventilatory support. The models were developed and cross-validated for ventilator-associated pneumonia, acute kidney injury, complicated dispositions, and return to the operating room, yielding predictive areas under the curve from 0.70 to 0.91. Each model derived its optimal predictive value by combining clinical, administrative, and immunological analyte data., Conclusion: Clinical, immunological, and administrative data can be combined to predict post-traumatic outcomes and resource use. Multidimensional machine learning modeling can identify trauma patients with complicated clinical trajectories and high resource needs., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objective: To design and establish a prospective biospecimen repository that integrates multi-omics assays with clinical data to study mechanisms of controlled injury and healing., Background: Elective surgery is an opportunity to understand both the systemic and focal responses accompanying controlled and well-characterized injury to the human body. The overarching goal of this ongoing project is to define stereotypical responses to surgical injury, with the translational purpose of identifying targetable pathways involved in healing and resilience, and variations indicative of aberrant peri-operative outcomes., Methods: Clinical data from the electronic medical record combined with large-scale biological data sets derived from blood, urine, fecal matter, and tissue samples are collected prospectively through the peri-operative period on patients undergoing 14 surgeries chosen to represent a range of injury locations and intensities. Specimens are subjected to genomic, transcriptomic, proteomic, and metabolomic assays to describe their genetic, metabolic, immunologic, and microbiome profiles, providing a multidimensional landscape of the human response to injury., Results: The highly multiplexed data generated includes changes in over 28,000 mRNA transcripts, 100 plasma metabolites, 200 urine metabolites, and 400 proteins over the longitudinal course of surgery and recovery. In our initial pilot dataset, we demonstrate the feasibility of collecting high quality multi-omic data at pre- and postoperative time points and are already seeing evidence of physiologic perturbation between timepoints., Conclusions: This repository allows for longitudinal, state-of-the-art geno-mic, transcriptomic, proteomic, metabolomic, immunologic, and clinical data collection and provides a rich and stable infrastructure on which to fuel further biomedical discovery., Competing Interests: The authors report no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Costimulation blockade (CoB)-based immunosuppression offers the promise of improved transplantation outcomes with reduced drug toxicity. However, it is hampered by early acute rejections, mediated at least in part by differentiated, CoB-resistant T cells, such as CD57 + PD1 - CD4 T cells. In this study, we characterize these cells pretransplant, determine their fate posttransplant, and examine their proliferative capacity in vitro in humans. Our studies show that CD57 + PD1 - CD4 T cells are correlated with increasing age and CMV infection pretransplant, and persist for up to 1 y posttransplant. These cells are replication incompetent alone but proliferated in the presence of unsorted PBMCs in a contact-independent manner. When stimulated, cells sorted by CD57/PD1 status upregulate markers of activation with proliferation. Up to 85% of CD57 + PD1 - cells change expression of CD57/PD1 with stimulation, typically, upregulating PD1 and downregulating CD57. PD1 upregulation is accentuated in the presence of rapamycin but prevented by tacrolimus. These data support a general theory of CoB-resistant cells as Ag-experienced, costimulation-independent cells and suggest a mechanism for the synergy of belatacept and rapamycin, with increased expression of the activation marker PD1 potentiating exhaustion of CoB-resistant cells., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Vascularized composite allotransplants (VCAs) seem to have several unique features of clinical and experimental importance, including uniquely definable lymphatic drainage that can be easily accessed at the level of ipsilateral regional node beds. Thus, VCA offers a unique opportunity to assess the relative contribution of peripheral and secondary lymphoid tissue to the process of rejection. We transplanted hind limb grafts from C3H donors to six different groups of C57BL/6 recipients: Spleen + Map3k14 -/- ; Spleen - Map3k14 -/- ; Spleen + Node - Map3k14 -/- ; and Spleen - Node - Map3k14 -/- . As positive controls, we used Map3k14 +/- with or without spleen. Map3k14 +/- mice demonstrated an average graft survival of 9.6 and 9.2 days for Spleen - and Spleen + Map3k14 +/- , respectively. Rejection in the Map3k14 -/- group was considerably delayed (28.4 days, P = 0.002) in all recipients. The Spleen - Map3k14 -/- mice rejected their hind limb allografts in an even more delayed fashion compared to Spleen + Map3k14 -/- (54.4 days, P = 0.02). Histological analysis of skin showed that acute rejection in both Map3k14 +/- mice groups was graded as Banff III or Banff IV. In the Map3k14 -/- groups, rejection was graded as Banff III. We demonstrated that in the absence of lymph nodes, grafts reject in a delayed fashion. Also, splenectomy in alymphoplastic mice further extends graft survival, but does not eliminate rejection all together., (© 2021 Steunstichting ESOT. Published by John Wiley & Sons Ltd.)

Depletional induction using antithymocyte globulin (ATG) reduces rates of acute rejection in adult kidney transplant recipients, yet little is known about its effects in children. Using a longitudinal cohort of 103 patients in the Immune Development in Pediatric Transplant (IMPACT) study, we compared T cell phenotypes after ATG or non-ATG induction. We examined the effects of ATG on the early clinical outcomes of alloimmune events (development of de novo donor specific antibody and/or biopsy proven rejection) and infection events (viremia/viral infections). Long-term patient and graft outcomes were examined using the Scientific Registry of Transplant Recipients. After ATG induction, although absolute counts of CD4 and CD8 T cells were lower, patients had higher percentages of CD4 and CD8 memory T cells with a concomitant decrease in frequency of naïve T cells compared to non-ATG induction. In adjusted and unadjusted models, ATG induction was associated with increased early event-free survival, with no difference in long-term patient or allograft survival. Decreased CD4 + naïve and increased CD4 + effector memory T cell frequencies were associated with improved clinical outcomes. Though immunologic parameters are drastically altered with ATG induction, long-term clinical benefits remain unclear in pediatric patients., (© 2020 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Vascularized composite allotransplantation (VCA) is challenged by the morbidity of immunosuppression required to prevent rejection. The use of highly specific biologics has not been well explored in VCA. Given that psoriasis is T-cell mediated, as is rejection of skin-containing VCAs, we sought to assess the role of ustekinumab and secukinumab, which are approved to treat psoriasis by inhibiting Th17 cells. We combined these agents with belatacept and steroids in a VCA nonhuman primate model. Group I consisted of belatacept and steroids, group II was belatacept, ustekinumab with steroid taper, and group III was belatacept, secukinumab with steroid taper. Three animals were transplanted in each group. In group I, the mean graft survival time until the first sign of rejection was 10 days whereas in group II and III it was 10.33 and 11 days, respectively. The immunohistochemistry analysis showed that the number of IL-17a + cells and the intensity of IL-17a expression were significantly reduced in both dermis and hypodermis parts in groups II and III when compared to group I (P < 0.01). Ustekinumab and secukinumab led to less T-cell infiltration and IL-17a expression in the allograft but provided no benefit to belatacept and steroids in VCA survival., (2020 Steunstichting ESOT. Published by John Wiley & Sons Ltd.)

Background and Aims: As conversion from calcineurin inhibitor to sirolimus (SRL), a mechanistic target of rapamycin inhibitor (mTOR-I), has been shown to enhance immunoregulatory profiles in liver transplant (LT) recipients (LTRs), mTOR-I therapy might allow for increased success of immunosuppression (IS) withdrawal. Our aim was to determine if operational tolerance could be observed in LTRs withdrawn from SRL and if blood/graft tolerance biomarkers were predictive of successful withdrawal., Approach and Results: We performed a prospective trial of SRL monotherapy withdrawal in nonimmune, nonviremic LTRs > 3 years post-LT. SRL was weaned over ~6 months, and biopsies were performed 12 months postweaning or at concern for acute rejection. Twenty-one LTRs consented; 6 were excluded due to subclinical acute rejection on baseline biopsy or other reasons, and 15 underwent weaning (age 61.3 ± 8.8 years; LT to SRL weaning 6.7 ± 3 years). Eight (53%) achieved operational tolerance (TOL). Of the 7 who were nontolerant (non-TOL), 6 had mild acute rejection on biopsy near the end of weaning or at study end; 1 was removed from the trial due to liver cancer recurrence. At baseline preweaning, there were statistically increased blood tolerogenic dendritic cells and cell phenotypes correlating with chronic antigen presentation in the TOL versus non-TOL groups. A previously identified biopsy gene signature accurately predicted TOL versus non-TOL in 12/14 LTRs before weaning. At study end, biopsy staining revealed statistically significant increases in antigen-presenting cell:leukocyte pairings, FOXP3 + /CD4 + T cells, Tbet + /CD8 + T cells, and lobular dendritic cells in the non-TOL group., Conclusions: This study evaluated IS withdrawal directly from mTOR-I therapy in LTRs and achieved > 50% operational tolerance. Preweaning gene expression and peripheral blood mononuclear cell profiling may be useful as predictors of successful mTOR-I therapy withdrawal. NCT02062944., (© 2019 by the American Association for the Study of Liver Diseases.)

This study evaluated the short-term effects of tofacitinib treatment on peripheral blood leukocyte phenotype and function, and the reversibility of any such effects following treatment withdrawal in healthy volunteers. Cytomegalovirus (CMV)-seropositive subjects received oral tofacitinib 10 mg twice daily for 4 weeks and were followed for 4 weeks after drug withdrawal. There were slight increases in total lymphocyte and total T-cell counts during tofacitinib treatment, and B-cell counts increased by up to 26%. There were no significant changes in granulocyte or monocyte counts, or granulocyte function. Naïve and central memory T-cell counts increased during treatment, while all subsets of activated T cells were decreased by up to 69%. T-cell subsets other than effector memory cluster of differentiation (CD)4+, activated naïve CD4+ and effector CD8+ T-cell counts and B-cell counts, normalized 4 weeks after withdrawal. Following ex vivo activation, measures of CMV-specific T-cell responses, and antigen non-specific T-cell-mediated cytotoxicity and interferon (IFN)-γ production, decreased slightly. These T-cell functional changes were most pronounced at Day 15, partially normalized while still on tofacitinib and returned to baseline after drug withdrawal. Total natural killer (NK)-cell counts decreased by 33%, returning towards baseline after drug withdrawal. NK-cell function decreased during tofacitinib treatment, but without a consistent time course across measured parameters. However, markers of NK-cell-mediated cytotoxicity, antibody-dependent cellular cytotoxicity and IFN-γ production were decreased up to 42% 1 month after drug withdrawal. CMV DNA was not detectable in whole blood, and there were no cases of herpes zoster reactivation. No new safety concerns arose. In conclusion, the effect of short-term tofacitinib treatment on leukocyte composition and function in healthy CMV+ volunteers is modest and largely reversible 4 weeks after withdrawal., (Copyright © 2018 Pfizer Inc. Published by Elsevier Inc. All rights reserved.)

An individual's immune function, susceptibility to infection, and response to immunosuppressive therapy are influenced in part by his/her T cell maturation state. Although childhood is the most dynamic period of immune maturation, scant information regarding the variability of T cell maturation in children with renal disease is available. In this study, we compared the T cell phenotype in children with renal failure (n=80) with that in healthy children (n=20) using multiparameter flow cytometry to detect markers of T cell maturation, exhaustion, and senescence known to influence immune function. We correlated data with the degree of renal failure (dialysis or nondialysis), prior immunosuppression use, and markers of inflammation (C-reactive protein and inflammatory cytokines) to assess the influence of these factors on T cell phenotype. Children with renal disease had highly variable and often markedly skewed maturation phenotypes, including CD4/CD8 ratio reversal, increased terminal effector differentiation in CD8 + T cells, reduction in the proportion of naïve T cells, evidence of T cell exhaustion and senescence, and variable loss of T cell CD28 expression. These findings were most significant in patients who had experienced major immune insults, particularly prior immunosuppressive drug exposure. In conclusion, children with renal disease have exceptional heterogeneity in the T cell repertoire. Cognizance of this heterogeneity might inform risk stratification with regard to the balance between infectious risk and response to immunosuppressive therapy, such as that required for autoimmune disease and transplantation., (Copyright © 2016 by the American Society of Nephrology.)

Graft-versus-host disease (GVHD) is the most common complication of hematopoietic stem cell transplant (HCT). However, our understanding of the molecular pathways that cause this disease remains incomplete, leading to inadequate treatment strategies. To address this, we measured the gene expression profile of nonhuman primate (NHP) T cells during acute GVHD. Utilizing microarray technology, we measured the expression profiles of CD3(+) T cells from five cohorts: allogeneic transplant recipients receiving (i) no immunoprophylaxis (No Rx), (ii) sirolimus monotherapy (Siro), (iii) tacrolimus-methotrexate (Tac-Mtx), as well as (iv) autologous transplant recipients (Auto) and (v) healthy controls (HC). This comparison allowed us to identify transcriptomic signatures specific for alloreactive T cells and determine the impact of both mTOR (mechanistic target of rapamycin) and calcineurin inhibition on GVHD. We found that the transcriptional profile of unprophylaxed GVHD was characterized by significant perturbation of pathways regulating T cell proliferation, effector function, and cytokine synthesis. Within these pathways, we discovered potentially druggable targets not previously implicated in GVHD, prominently including aurora kinase A (AURKA). Utilizing a murine GVHD model, we demonstrated that pharmacologic inhibition of AURKA could improve survival. Moreover, we found enrichment of AURKA transcripts both in allo-proliferating T cells and in sorted T cells from patients with clinical GVHD. These data provide a comprehensive elucidation of the T cell transcriptome in primate acute GVHD and suggest that AURKA should be considered a target for preventing GVHD, which, given the many available AURKA inhibitors in clinical development, could be quickly deployed for the prevention of GVHD., (Copyright © 2015, American Association for the Advancement of Science.)

Although cytomegalovirus (CMV) reactivation has long been implicated in posttransplant immune dysfunction, the molecular mechanisms that drive this phenomenon remain undetermined. To address this, we combined multiparameter flow cytometric analysis and T-cell subpopulation sorting with high-throughput sequencing of the T-cell repertoire, to produce a thorough evaluation of the impact of CMV reactivation on T-cell reconstitution after unrelated-donor hematopoietic stem cell transplant. We observed that CMV reactivation drove a >50-fold specific expansion of Granzyme B(high)/CD28(low)/CD57(high)/CD8(+) effector memory T cells (Tem) and resulted in a linked contraction of all naive T cells, including CD31(+)/CD4(+) putative thymic emigrants. T-cell receptor β (TCRβ) deep sequencing revealed a striking contraction of CD8(+) Tem diversity due to CMV-specific clonal expansions in reactivating patients. In addition to querying the topography of the expanding CMV-specific T-cell clones, deep sequencing allowed us, for the first time, to exhaustively evaluate the underlying TCR repertoire. Our results reveal new evidence for significant defects in the underlying CD8 Tem TCR repertoire in patients who reactivate CMV, providing the first molecular evidence that, in addition to driving expansion of virus-specific cells, CMV reactivation has a detrimental impact on the integrity and heterogeneity of the rest of the T-cell repertoire. This trial was registered at www.clinicaltrials.gov as #NCT01012492., (© 2015 by The American Society of Hematology.)