CD16+ natural killer cells in bronchoalveolar lavage are associated with antibody-mediated rejection and chronic lung allograft dysfunction.

Acute and chronic rejections limit the long-term survival after lung transplant. Pulmonary antibody-mediated rejection (AMR) is an incompletely understood driver of long-term outcomes characterized by donor-specific antibodies (DSAs), innate immune infiltration, and evidence of complement activation. Natural killer (NK) cells may recognize DSAs via the CD16 receptor, but this complement-independent mechanism of injury has not been explored in pulmonary AMR. CD16+ NK cells were quantified in 508 prospectively collected bronchoalveolar lavage fluid samples from 195 lung transplant recipients. Associations between CD16+ NK cells and human leukocyte antigen mismatches, DSAs, and AMR grade were assessed by linear models adjusted for participant characteristics and repeat measures. Cox proportional hazards models were used to assess CD16+ NK cell association with chronic lung allograft dysfunction and survival. Bronchoalveolar lavage fluid CD16+ NK cell frequency was associated with increasing human leukocyte antigens mismatches and increased AMR grade. Although NK frequencies were similar between DSA+ and DSA- recipients, CD16+ NK cell frequencies were greater in recipients with AMR and those with concomitant allograft dysfunction. CD16+ NK cells were associated with long-term graft dysfunction after AMR and decreased chronic lung allograft dysfunction-free survival. These data support the role of CD16+ NK cells in pulmonary AMR.

Gene signatures common to allograft rejection are associated with lymphocytic bronchitis.

Lymphocytic bronchitis (LB) precedes chronic lung allograft dysfunction. The relationships of LB (classified here as Endobronchial or E-grade rejection) to small airway (A- and B-grade) pathologies are unclear. We hypothesized that gene signatures common to allograft rejection would be present in LB. We studied LB in two partially overlapping lung transplant recipient cohorts: Cohort 1 included large airway brushes (6 LB cases and 18 post-transplant referents). Differential expression using DESeq2 was used for pathway analysis and to define an LB-associated metagene. In Cohort 2, eight biopsies for each pathology subtype were matched with pathology-free biopsies from the same subject (totaling 48 samples from 24 subjects). These biopsies were analyzed by multiplexed digital counting of immune transcripts. Metagene score differences were compared by paired t tests. Compared to referents in Cohort 1, LB demonstrated upregulation of allograft rejection pathways, and upregulated genes in these cases characterized an LB-associated metagene. We observed statistically increased expression in Cohort 2 for this LB-associated metagene and four other established allograft rejection metagenes in rejection vs paired non-rejection biopsies for both E-grade and A-grade subtypes, but not B-grade pathology. Gene expression-based categorization of allograft rejection may prove useful in monitoring lung allograft health.

Suppressed calcineurin-dependent gene expression identifies lung allograft recipients at increased risk of infection.

Lung transplant immunosuppression regimens generally include the calcineurin inhibitor tacrolimus. We hypothesized that mean residual expression (MRE) of calcineurin-dependent genes assesses rejection and infection risk better than does tacrolimus trough. We prospectively followed 44 lung allograft recipients at 2 to 18 months posttransplant and measured changes in whole blood interleukin-2, interferon-γ, and granulocyte-macrophage colony-stimulating factor gene expression following a tacrolimus dose. Posttransplant duration, immunosuppressive medication levels, and bronchoscopic rejection and infection assessments were compared with MRE by using generalized-estimating equation-adjusted models. Prednisolone effect on MRE was assessed ex vivo in blood samples from nontransplanted controls. Tacrolimus concentration inhibiting 50% of cytokine production (IC50 ) was measured in a pretransplant subset. Results showed that MRE did not change with diagnosis of rejection but that airway infection was associated with a 20% absolute decrease (95% confidence interval 11%-29%). MRE increased with time after transplant but was not associated with tacrolimus trough. Interestingly, MRE correlated inversely with corticosteroid dose in the study cohort and ex vivo. Pretransplant tacrolimus IC50 depended on the cytokine measured and varied between individuals, suggesting a range in baseline responses to tacrolimus. We conclude that MRE identifies infection risk in lung allograft recipients, potentially integrating calcineurin inhibitor and steroid effects on lymphocyte effector function.

Genotypes associated with tacrolimus pharmacokinetics impact clinical outcomes in lung transplant recipients.

Most lung transplantation immunosuppression regimens include tacrolimus. Single nucleotide polymorphisms (SNPs) in genes important to tacrolimus bioavailability and clearance (ABCB1, CYP3A4, and CYP3A5) are associated with differences in tacrolimus pharmacokinetics. We hypothesized that polymorphisms in these genes would impact immunosuppression-related outcomes. We categorized ABCB1, CYP3A4, and CYP3A5 SNPs for 321 lung allograft recipients. Genotype effects on time to therapeutic tacrolimus level, interactions with antifungal medications, concentration to dose (C0 /D), acute kidney injury, and rejection were assessed using linear models adjusted for subject characteristics and repeat measures. Compared with CYP3A poor metabolizers (PM), time to therapeutic tacrolimus trough was increased by 5.1 ± 1.6 days for CYP3A extensive metabolizers (EM, P < 0.001). In the post-operative period, CYP3A intermediate metabolizers spent 1.2 ± 0.5 days less (P = 0.01) and EM spent 2.1 ± 0.5 days less (P < 0.001) in goal tacrolimus range than CYP3A PM. Azole antifungals interacted with CYP3A genotype in predicting C0 /D (P < 0.001). Increased acute kidney injury rates were observed in subjects with high ABCB1 function (OR 3.0, 95% CI 1.1-8.6, P = 0.01). Lower rates of acute cellular rejection were observed in subjects with low ABCB1 function (OR 0.36, 95% CI 0.07-0.94, P = 0.02). Recipient genotyping may help inform tacrolimus dosing decisions and risk of adverse clinical outcomes.

Community-associated CMRSA-10 (USA-300) is the predominant strain among methicillin-resistant Staphylococcus aureus strains causing skin and soft tissue infections in patients presenting to the emergency department of a Canadian tertiary care hospital.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emerging pathogen first described among individuals with no contact with health care facilities. The purpose of this study was to determine the proportion of CA-MRSA, defined by pulsed field gel electrophoresis (PFGE), in MRSA skin and soft tissue infections presenting to the Emergency Department (ED). We also aimed to describe the laboratory and clinical characteristics of CA-MRSA infections. From June 1, 2001 to May 30, 2005, MRSA isolates from skin and soft tissue infections presenting to the ED were reviewed. They were characterized by antibiotic susceptibilities and PFGE, and the presence of staphylococcal cassette chromosome (SCC) mec type IVa and Panton-Valentine leukocidin (PVL) genes was assessed on representative isolates. The medical records were reviewed to define risk factors. There were 95 isolates available for analysis, of which 58 (61%) were CMRSA-10 (USA-300), the predominant clone from 2003 onward. All representative isolates (24%) tested in this group had PVL genes and SCCmec type IVa. Their antibiogram showed 100% susceptibility to trimethoprim-sulfamethoxazole, rifampin, and fusidic acid, and 79% to clindamycin. Clinical comparison of CMRSA-10 vs. hospital PFGE type strains showed 22% vs. 60%, respectively, for recent antibiotic use (p < 0.0001), 26% vs. 6%, respectively, for intravenous drug use (p < 0.05), and 57% vs. 6%, respectively, for soft tissue abscess (p < 0.001). CMRSA-10 is a major pathogen in skin and soft tissue abscesses in our ED. It has a characteristic susceptibility, and was associated with intravenous drug use, but not with recent antibiotic usage.

Dectin-1 genetic deficiency predicts chronic lung allograft dysfunction and death.

BACKGROUNDInnate immune activation impacts lung transplant outcomes. Dectin-1 is an innate receptor important for pathogen recognition. We hypothesized that genotypes reducing dectin-1 activity would be associated with infection, graft dysfunction, and death in lung transplant recipients.METHODSWe assessed the rs16910526 CLEC7A gene polymorphism Y238X, which results in dectin-1 truncation, in 321 lung allograft recipients at a single institution and in 1,129 lung allograft recipients in the multicenter Lung Transplant Outcomes Group (LTOG) cohort. Differences in dectin-1 mRNA, cytokines, protein levels, immunophenotypes, and clinical factors were assessed.RESULTSY238X carriers had decreased dectin-1 mRNA expression (P = 0.0001), decreased soluble dectin-1 protein concentrations in bronchoalveolar lavage (P = 0.008) and plasma (P = 0.04), and decreased monocyte surface dectin-1 (P = 0.01) compared with wild-type subjects. Y238X carriers had an increased risk of fungal pathogens (HR 1.17, CI 1.0-1.4), an increased risk of graft dysfunction or death (HR 1.6, CI 1.0-2.6), as well increased mortality in the UCSF cohort (HR 1.8, CI 1.1-3.8) and in the LTOG cohort (HR 1.3, CI 1.1-1.6), compared with wild-type CLEC7A subjects.CONCLUSIONIncreased rates of graft dysfunction and death associated with this dectin-1 polymorphism may be amplified by immunosuppression that drives higher fungal burden from compromised pathogen recognition.FUNDINGThe UCSF Nina Ireland Program for Lung Health Innovative Grant program, the Clinical Sciences Research & Development Service of the VA Office of Research and Development, and the Joel D. Cooper Career Development Award from the International Society for Heart and Lung Transplantation.

NKG2C Natural Killer Cells in Bronchoalveolar Lavage Are Associated With Cytomegalovirus Viremia and Poor Outcomes in Lung Allograft Recipients.

BACKGROUND: Cytomegalovirus (CMV) infection is a risk factor for chronic lung allograft dysfunction (CLAD), which limits survival in lung allograft recipients. Natural killer (NK) cells that express the NKG2C receptor mediate CMV-specific immune responses. We hypothesized that NKG2C NK cells responding to CMV in the lung allograft would reduce CMV-related inflammation and would improve CLAD-free survival. METHODS: We prospectively followed 130 subjects who underwent lung transplantation from 2012 to 2016. Bronchoalveolar lavage (BAL) NK cells were immunophenotyped for NKG2C, maturation, and proliferation markers. CMV viral load, serologies, serial spirometry, and mortality were recorded from medical records. Natural killer cell subset association with CMV endpoints were made using generalized estimating equation-adjusted linear models. BAL NKG2C NK cell association with CLAD-free survival was assessed by Cox proportional hazards modeling. RESULTS: NKG2C NK cells were more mature and proliferative than NKG2C NK cells and represented a median of 7.8% of BAL NK cells. The NKG2C NK cell proportion increased prior to the first detection of viremia and was nearly tripled in subjects with high level viremia (>1000 copies/mL) compared with no detected viremia. Subjects with increased BAL NKG2C NK cells, relative to the median, had a significantly increased risk for CLAD or death (hazard ratio, 4.2; 95% confidence interval, 1.2-13.3). CONCLUSIONS: The BAL NKG2C NK cell proportion may be a relevant biomarker for assessing risk of CMV viremia and quantifying potential CMV-related graft injury that can lead to CLAD or death.

Tacrolimus trough monitoring guided by mass spectrometry without accounting for assay differences is associated with acute kidney injury in lung transplant recipients.

PURPOSE: Tacrolimus is a nephrotoxic immunosuppressant historically monitored via enzyme-based immunoassay (IA). After 2011, the 2 largest laboratory companies in the United States implemented tacrolimus quantification by liquid chromatography-mass spectrometry (LC-MS); this method excludes metabolites, potentially resulting in lower quantified drug concentrations. We sought to determine if tacrolimus therapeutic drug monitoring via LC-MS, as performed using trough targets originally derived from IA values, influences clinical outcomes. METHODS: In a single-center retrospective cohort study of lung transplant recipients, risks of acute kidney injury, acute renal failure, and new-onset diabetes after transplantation, as well as chronic lung allograft dysfunction-free survival, were compared in 82 subjects monitored by LC-MS and 102 subjects monitored by IA using Cox proportional hazard models adjusted for age, sex, baseline renal function, and race. RESULTS: LC-MS-based monitoring was associated with a greater risk of acute kidney injury (adjusted hazard ratio, 1.65; 95% confidence interval, 1.02-2.67). No statistically significant differences in risks of acute renal failure and new-onset diabetes after transplantation were observed. CONCLUSION: Although LC-MS provides a more accurate representation of the blood concentration of the parent compound tacrolimus exclusive of metabolite, established cut points for tacrolimus dosing may need to be adjusted to account for the increased risk of renal injury.